Study of Actinobacteria and Their Secondary Metabolites from Various Habitats in Indonesia and Deep-Sea of the North Atlantic Ocean

Study of Actinobacteria and their Secondary Metabolites from Various Habitats in Indonesia and Deep-Sea of the North Atlantic Ocean

Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina zu Braunschweig

zur Erlangung des Grades eines

Doktors der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n

von Chandra Risdian aus Jakarta / Indonesien

1. Referent: Professor Dr. Michael Steinert 2. Referent: Privatdozent Dr. Joachim M. Wink eingereicht am: 18.12.2019 mündliche Prüfung (Disputation) am: 04.03.2020

Druckjahr 2020 ii

Vorveröffentlichungen der Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht:

Publikationen

Risdian C, Primahana G, Mozef T, Dewi RT, Ratnakomala S, Lisdiyanti P, and Wink J. Screening of antimicrobial producing Actinobacteria from Enggano Island, Indonesia. AIP Conf Proc 2024(1):020039 (2018).

Risdian C, Mozef T, and Wink J. Biosynthesis of polyketides in Streptomyces. Microorganisms 7(5):124 (2019)

Posterbeiträge

Risdian C, Mozef T, Dewi RT, Primahana G, Lisdiyanti P, Ratnakomala S, Sudarman E, Steinert M, and Wink J. Isolation, characterization, and screening of antibiotic producing Streptomyces spp. collected from soil of Enggano Island, Indonesia. The 7th HIPS Symposium, Saarbrücken, Germany (2017). Risdian C, Ratnakomala S, Lisdiyanti P, Mozef T, and Wink J. Multilocus sequence analysis of Streptomyces sp. SHP 1-2 and related species for phylogenetic and taxonomic studies. The HIPS Symposium, Saarbrücken, Germany (2019).

iii

Acknowledgements

First and foremost I would like to express my deep gratitude to my mentor PD Dr. Joachim Wink for giving me the privilege to do my PhD in his working group Microbial Strain Collection at the HZI. I thank him for his support, encouragement and guidance for carrying out my work.

I would like to offer my great appreciation to Prof. Dr. Marc Stadler for his support, suggestion, and giving the opportunity to work in his laboratories in Microbial Drugs group. I also want to thank Prof. Dr. Michael Steinert for giving me opportunity as a PhD student in TU Braunschweig and becoming one of my thesis committee members.

My deepest thanks to Dr. Tjandrawati Mozef as an Indonesian counterpart project leader of GINAICO (German-Indonesian Anti-Infective Cooperation). I want to thank Dr. Puspita Lisdiyanti, Dr. Shanti Ratnakomala, and Dr. Rizna Triana Dewi and all of my Indonesian collaboration partners from Research Center for Chemistry and Research Center for Biotechnology LIPI.

I would like to give special thanks to the DAAD (Deutscher Akademischer Austauschdienst) for the financial support and assistance during my PhD studies, and to German Federal Ministry of Education and Research (BMBF) under the GINAICO project, the President’s Initiative and Networking Funds of the Helmholtz Association of German Research Centres under Contract Number VHGS-202, and Ministry of Research, Technology and Higher Education of the Republic of Indonesia (RISTEKDIKTI) for supporting my PhD project.

I extend my gratitudes to my collaboration partners Prof. Dr. Eike Steinmann, Dr. Kathrin Mohr, Dr. Sabrina Karwehl, Dr. Enge Sudarman, Dr. Rolf Jansen, Dr. Manfred Rohde, Dr. Richard Hahnke, Dr. Peter Schumann, Dr. Cathrin Spröer, Dr. Boyke Bunk, and Gabriele Pötter.

I would like to thank members of the working groups Microbial Strain Collection especially Christiane Fritz-Braun, Kerstin Schober, Silke Reinecke, Axel Schulz and Tian Cheng for the great support during my PhD work. I want to thank Gian Primahana, Senlie Octaviana, Zahra Noviana, and Dimas F. Praditya for helping me in chemistry part, Actinobacteria isolation, DNA analysis, and antiviral assay, and TU iv

Acknowledgements

Braunschweig students from IB20B practical group.

My deepest thanks to Romy Schade, Aileen Gollasch, Klaus Peter Conrad, Stephanie Schulz, Birte Trunkwalter, Wera Collisi, Jolanta Lulla, Hannes Meyer for the best technical support. I thank all of my other colleagues in the Microbial Strain Collection at the HZI for the good work environment: Dr. Lucky Mulwa, Rina Andriyani, Shadi Khodamoradi, and Nasim Safaei.

Lastly, I would like to dedicate this work to my family for their love and support throughout the years, especially to my wife Yayu Sri Rahayu and my children Nashwa Levareyna Risdian and Naufal Zeinizar Risdian.

Table of Contents

Acknowledgements ...... iv Table of Contents ...... vi List of Figures ...... ix List of Tables ...... xi List of Abbreviations ...... xii Abstract ...... xiii Abstrakt ...... xiv 1 Introduction ...... 1 1.1 Natural product and drug discovery ...... 1 1.2 Actinobacteria ...... 5 1.2.1 Streptomyces ...... 7 1.2.2 Non-Streptomyces Actinobacteria ...... 9 1.3 Polyphasic taxonomy of the class Actinobacteria ...... 11 1.3.1 Phenotypic analysis ...... 14 1.3.2 Genomic and phylogenetic analysis ...... 16 1.4 Dereplication and isolation of natural products from Actinobacteria ..... 18 1.5 Hepatitis ...... 19 1.6 Previous work ...... 20 1.7 Aim of study...... 20 2 Materials and Methods ...... 21 2.1 Materials...... 21 2.1.1 Media ...... 21 2.1.2 Chemicals ...... 25 2.1.3 Source of samples ...... 27 2.1.4 Organisms ...... 30 2.1.5 Kits and enzymes ...... 32 2.1.6 Primers ...... 32 2.1.7 Equipment ...... 34 2.2 Methods ...... 35 2.2.1 Sampling ...... 35 2.2.2 Isolation of Actinobacteria ...... 35 2.2.3 Extract Production...... 35 2.2.4 Analysis of 16S rRNA sequences ...... 36 vi

Table of Contents

2.2.5 Morphology and melanin production ...... 37 2.2.6 Physiological and biochemical characteristic ...... 37 2.2.6.1 Growth at different temperature and pH ...... 37 2.2.6.2 Carbon utilization ...... 38 2.2.6.3 Sodium chloride tolerance ...... 38 2.2.6.4 Enzymatic assay ...... 38 2.2.6.5 Antibiotic susceptibility ...... 39 2.2.7 Chemotaxonomy ...... 39 2.2.7.1 Freeze-dried cells preparation ...... 39 2.2.7.2 Cell wall amino acid analysis ...... 39 2.2.7.3 Whole-cell sugar analysis ...... 40 2.2.7.4 Menaquinone analysis...... 40 2.2.7.5 Polar lipid analysis ...... 41 2.2.7.6 Fatty acid analysis ...... 41 2.2.8 G+C (guanine + cytosine) contents determination ...... 42 2.2.9 Multilocus sequence analysis (MLSA) for Streptomyces sp. SHP 1-2. .. 43 2.2.10 Bioactivity assay ...... 44 2.2.10.1 Antimicrobial assay ...... 44 2.2.10.2 Nematicidal actitity ...... 44 2.2.10.3 Cytotoxic assay ...... 45 2.2.10.4 Antiviral assay ...... 45 2.2.11 Fermentation, extraction and isolation of secondary metabolites from Streptomyces sp. SHP 1-2 ...... 46 2.2.12 Fermentation, extraction and isolation of compound from Amycolatopsis sp. 196526CR ...... 46 3 Results ...... 48 3.1 Isolated Actinobacteria, 16S rRNA gene characterization, and bioactivity of the extracts ...... 48 3.2 Polyphasic taxonomy of Streptomyces sp. SHP 1-2 ...... 50 3.2.1 16S rRNA gene analysis ...... 52 3.2.2 Multilocus sequence analysis (MLSA) ...... 53 3.2.3 G+C content and full genome sequencing ...... 55 3.2.4 Physiological and biochemical characteristic ...... 55 3.2.5 Phenotypic characteristic between Streptomyces sp. SHP 1-2 and its closest related strains ...... 58

vii

Table of Contents

3.3 Compounds isolated from Streptomyces sp. SHP 1-2 and their biological activity ...... 60 3.4 Taxonomy study and extract analysis of strain MAE 1-11 ...... 62 3.4.1 Morphology and melanin production ...... 62 3.4.2 16S rRNA gene analysis ...... 62 3.4.3 Physiological and biochemical characteristic ...... 64 3.4.4 Raw extract analysis...... 65 3.5 Taxonomic study of strain 196526CR ...... 65 3.5.1 Morphology and melanin production ...... 65 3.5.2 16S rRNA gene analysis ...... 65 3.5.3 Physiological and biochemical characteristic ...... 69 3.5.4 Secondary metabolites produced by strain 196526CR ...... 69 3.6 Taxonomic study of Streptomyces sp. ASO4wet ...... 73 3.6.1 Morphology and physiology ...... 73 3.6.2 Chemotaxonomy ...... 74 3.6.3 16S rRNA gene analysis ...... 74 3.6.4 Full genome analysis ...... 75 4 Discussion ...... 79 4.1 Isolated Actinobacteria and their bioactivity ...... 79 4.2 Description of strain SHP 1-2 and its secondary metabolites ...... 80 4.3 Description of strain MAE 1-11 and its secondary metabolites ...... 82 4.4 Description of strain 196526CR and its secondary metabolites ...... 84 4.5 Description of strain ASO4wet ...... 86 5 Summary ...... 88 6 References ...... 89 7 Appendix ...... 108

viii

List of Figures

Figure 1. Distribution of natural products produced by organisms ...... 1 Figure 2. Timeline of introduced antibiotics...... 3 Figure 3. Streptomyces sp. strain C196921 in agar medium ...... 8 Figure 4. Some non-Streptomyces strains in agar medium ...... 10 Figure 5. Map of Indonesia with the sampling sites...... 36 Figure 6. Morphology of Streptomyces sp. SHP 1-2 on GYM (left) and ISP 2 medium (right) ...... 51 Figure 7. Scanning electron micrographs of aerial mycelium and spores of Streptomyces sp. SHP 1-2 after incubation on ISP 3 agar for 14 days at 30 °C ...... 51 Figure 8. TLC chromatogram of whole-cell sugar analysis (left) and analysis amino acid of the cell wall (right) of Streptomyces sp. SHP 1-2 ...... 52 Figure 9. GC chromatogram of fatty acid analysis of Streptomyces sp. SHP 1-2 ..... 53 Figure 10. Menaquinones detected in Streptomyces sp. SHP 1-2...... 54 Figure 11. Polar lipid observed in Streptomyces sp. SHP 1-2...... 54 Figure 12. Neighbour-joining tree based on 16S rRNA gene sequences (1435 positions in the final dataset) showing relationships between strain SHP 1-2T and the type strains of closely related Streptomyces species...... 55 Figure 13. Neighbour-joining tree based on concatenated partial sequences of the house-keeping genes atpD, gyrB, recA, rpoB and trpB...... 56 Figure 14. G+C content from the genome of Streptomyces sp. SHP 1-2 based on HPLC anlaysis ...... 57 Figure 15. Compounds isolated from Streptomyces sp. SHP 1-2 ...... 60 Figure 16. Morphology of strain MAE 1-11 on GYM (left) and ISP 2 medium (right)...... 63 Figure 17. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences between strain MAE 1-11 and its closely related neighbours ...... 63 Figure 18. LC-HRMS analysis result of raw extract of strain MAE 1-11 produced by SYP medium...... 66 Figure 19. LC-HRMS analysis result of Bafilomycin D...... 67 Figure 20. Morphology of strain 196526CR on GYM (left) and ISP 2 medium (right)...... 68 Figure 21. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences between strain 196526CR and its closely related neighbours ...... 69 Figure 22. Structure of secondary metabolites produced by by strain 196526CR ..... 71 Figure 23. Chromatogram, UV and MS spectrum of Coproporphyrin III and Zn- Coproporphyrin III found in the XAD extract of strain 196526CR...... 72 Figure 24. Chromatogram, UV and MS spectrum of Nitrosoxacin C found in the biomass extract of strain 196526CR ...... 73

List of Figures

Figure 25. Scanning electron micrographs of aerial mycelium with no spore detected of strain ASO4wetT after incubation on ISP 3 agar for 4 weeks at 30°C ...... 74 Figure 26. TLC chromatogram of whole-cell sugar analysis (left) and analysis amino acid of the cell wall (right) of isolate ASO4wet ...... 75 Figure 27. GC chromatogram of fatty acid analysis of isolate ASO4wet...... 76 Figure 28. Menaquinones detected in isolate ASO4wet...... 77 Figure 29. Polar lipid observed in strain ASO4wet...... 77 Figure 30. Neighbour-joining tree based on 16S rRNA gene sequences (1408 positions in the final dataset) showing relationships between Streptomyces sp. ASO4wetT and its closely related Streptomyces species ...... 78

List of Tables

Table 1. Current taxonomic outline for the phylum Actinobacteria ...... 6 Table 2. Some antibiotics produced by Streptomyces species ...... 8 Table 3. Some antibiotics produced by non-Streptomyces species ...... 11 Table 4. List of media used in this study ...... 21 Table 5. List of chemicals used in this study ...... 25 Table 6. List of samples collected from Indonesia for Actinobacteria isolation ...... 27 Table 7. Actinobacteria strains from Research Center for Biotechnology LIPI- Indonesia ...... 30 Table 8. Tested organisms for antimicrobial, antinematode, and cytotoxic activity . 31 Table 9. The list of kits and enzymes used in the experiment ...... 32 Table 10. The list of primers used for 16S rRNA analysis ...... 32 Table 11. The list of primers used for multi locus sequence analysis or MLSA ...... 33 Table 12. Equipments used in this work ...... 34 Table 13. The activities that can be analyzed by API ZYM and API Coryne ...... 38 Table 14. Some isolated Actinobacteria with low percentage similarity from the closest species based on 16S rRNA gene ...... 48 Table 15. Selected strains that can produce extracts with moderate and strong activity against some microbes ...... 49 Table 16. List of non-toxic extracts having antiviral activity with strong level against HCV ...... 50 Table 17. Growth and characteristics of Streptomyces sp. SHP 1-2 cultivated on various agar media after incubation for 14 days at 30 °C ...... 50 Table 18. MLSA distance between Streptomyces sp. SHP 1-2 and other related strains ...... 57 Table 19. API ZYM and API Coryne test result for Streptomyces sp. SHP 1-2 ...... 58 Table 20. Some phenotypic comparison between Streptomyces sp. SHP 1-2 and its closest related strains...... 59 Table 21. Biological activity of compounds isolated from strain SHP 1-2 ...... 61 Table 22. Growth and characteristics of strain MAE 1-11 grown on various agar media after incubation for 14 days at 30°C ...... 62 Table 23. Some physiology properties of strain MAE 1-11 ...... 64 Table 24. Growth and characteristics of isolate 196526CR grown on various agar media after incubation for 14 days at 30°C ...... 68 Table 25. Some physiology properties of strain 196526CR ...... 70

List of Tables

List of Abbreviations

°C Degree Celsius 16S rRNA Component of the 30S small subunit of a prokaryotic ribosomal ribonucleic acid [M+H]+ Protonated molecular ion ASW Artificial sea water DAD Diode array detector DAP 2, 6-Diaminopimelic acid DDH DNA-DNA hybridization DNA Deoxyribonucleic acid DNP Dictionary of natural products EDTA Ethylenediaminetetraacetic acid G+C Guanine and cytosine HCV Hepatitis C virus HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High performance liquid chromatography HR-ESI-MS High resolution electron spray ionization mass spectrometry ISP International Streptomyces project JSRM Jump start ready mix LC-MS Liquid chromatography – mass spectrometry MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight MIC Minimal inhibition concentration MLSA Multilocus sequence analysis NMR Nuclear magnetic resonance NRPS Nonribosomal peptide synthetases OD Optical density OSMAC One strain-many compounds PCR Polymerase chain reaction PKS Polyketide synthases RNA Ribonucleic acid rpm Revolutions per minute

xii

Abstract

Actinobacteria are Gram-positive bacteria and the prolific producers of bioactive molecules. In the course of drug discovery from actinobacterial sources, some samples were taken from different locations and habitats of Indonesia and isolation of Actinobacteria was conducted thereof. Totally 196 strains of Actinobacteria were isolated, and approximately half of them were characterized by 16S rRNA gene analysis. Around two-thirds of them were detected as Streptomyces. One of the Streptomyces strains, i.e., strain SHP 1-2 produced active extracts against some microbial pathogens. Analysis and isolation of compounds from its extract revealed that two novel molecules, which are indolactam derivatives, were found from it. Polyphasic taxonomy study of strain SHP 1-2 suggested that this strain is a novel species in the Streptomyces group. Another study from different strains suggested that strain MAE-11, isolated from mangrove area, was identified close to Kitasatopora and Streptomyces species and generated active extract against hepatitis C virus (HCV). The extract was known to contain bafilomycin D as the molecule that was responsible for the antiviral activity. Isolate 196526CR was detected as Amycolatopsis species and could produce an extract that showed activity against some tested microbes. The compound analysis and isolation from its extract indicated that nitrosoxacin C possesses property as an antimicrobial compound. Chemotaxonomy study of strain ASO4wet, which was previously isolated from deep-sea in the North Atlantic Ocean, was also conducted and the data supported that ASO4wet belongs to the genus Streptomyces. The strain is described as the novel species.

Keywords: Actinobacteria, Streptomyces, drug discovery, Indonesia, deep sea

xiii

Abstrakt

Actinobakterien sind Gram-positive Bakterien und die ergiebigsten Produzenten bioaktiver Moleküle. Im Zuge der Wirkstoffforschung aus Actinobakterien wurden einige Proben an verschiedenen Orten und Lebensräumen Indonesiens entnommen und daraus Actinobakterien isoliert. Insgesamt wurden 196 Aktinobakterien-Stämme isoliert und etwa die Hälfte von ihnen wurde durch 16S-rRNA-Genanalyse charakterisiert. Etwa zwei Drittel von ihnen wurden als Streptomyces identifiziert. Einer der Streptomyces-Stämme, d. h. Stamm SHP 1-2, produzierte aktive Extrakte gegen einige pathogene Mikroorganismen. Nach Analyse und anschließender Isolierung der Inhaltsstoffe konnten zwei neue Substanzen, die zu der Indolactam- Derivaten gehören, gefunden werden. Eine polyphasische Taxonomiestudie des Stammes SHP 1-2 legte nahe, dass dieser Stamm zu einer neuen Spezies in der Gattung Streptomyces-gehört. Eine vergleichende Studie mit verschiedenen Stämmen legte nahe, dass der Stamm MAE-11, der aus Mangrovengebieten isoliert wurde, nahe verwandt mit Kitasatopora- und Streptomyces-Arten ist und einen aktiven Extrakt gegen das Hepatitis C-Virus (HCV) produziert. Es zeigte sich, dass der Extrakt Bafilomycin D als die Substanz enthielt, die für die antivirale Aktivität verantwortlich war. Das Isolat 196526CR wurde als Amycolatopsis-Spezies identifiziert und bildet einen Extrakt, der eine Aktivität gegen einige Mikroorganismen zeigte. Die Analyse der Bestandteile und die Isolierung aus diesem Extrakt zeigten, dass Nitrosoxacin C für die antimikrobielle Eigenschafte verantwortlich ist. Eine chemotaxonomiche Studie des Stammes ASO4wet, der aus der Tiefsee im Nordatlantik isoliert worden war, wurde ebenfalls durchgeführt, und die Daten untermauerten, dass ASO4wet zur Gattung Streptomyces gehört. Der Stamm wird als neue Art beschrieben.

Schlüsselwörter: Aktinobakterien, Streptomyces, Wirkstoffforschung, Indonesien, Tiefsee

xiv

1 Introduction

1 Introduction 1.1 Natural product and drug discovery Natural products are various chemical compounds produced by bacteria, fungi, plants, and animals. These include the primary metabolites such as DNA, RNA, and protein and the secondary metabolites such as isoprenoids, alkaloids, polyketides, and peptides, especially nonribosomal peptides1–4. However, the definition of natural product usually is used by many scientists to refer to the secondary metabolites produced by organisms1. Not like the primary metabolites, which are very important for the growth and reproduction, the secondary metabolites give other bioactivity benefits to the producing organisms. They could be used for defensive or attack mechanisms, competition with the other organisms, and interspecies communication. Therefore, it can be considered that the secondary metabolites are required for survival of the hosts in their environment5.

More than one million of natural products have been isolated recently. They are essential and valuable agents in human life. People have used them for many applications such as in nutrition, medicine and agriculture. Many of them originate from plants (50-60%) such as alkaloids, flavonoids, terpenoids, and steroids and microbes produce only a small portion (about 5%). However, only 20-25% of these compounds possess biological activity and circa 10% of these active molecules are produced by microbes, especially from Actinobacteria which contribute around 45% followed by fungi (~38%)6–8 as shown in Figure 1.

700000 Number of microbial bioactive 600000 natural products 500000 400000 300000 3800 200000 10100 8600 100000

0 Numbernatural of products Plants Animals Microbes

Bioactives Non-Bioactives Actinobacteria Other bacteria Fungi

Figure 1. Distribution of natural products produced by organisms. Data are derived from Bérdy 20058.

1 Introduction

Microbes have supplied compounds they produced that have helped people to overcome diseases for 70 years. These comprise antibiotics, antitumor drugs, immunosuppressants, enzyme inhibitors, and antiviral agents2. The history of drug discovery from microbial sources has been started since 1929 when Alexander Fleming published his work about the antimicrobial activity from the mould Penicillium notatum that killed Staphylococcus aureus9. Since then, many scientists have worked to find drugs from microbial sources. In 1942, Waksman and Tishler published their finding of actinomycin which was produced by Actinomyces antibioticus (the current name is Streptomyces antibioticus) that has antibacterial activity10.

The “heroic” or “golden era” of antibiotics is between the 1940s-1950s when almost all valuable antibacterial drugs were found, such as tetracyclines, cephalosporins, aminoglycosides, and macrolides. It was suggested that in the period between the 1950s-1960s, the major issues in chemotherapy had been resolved. Interestingly, antibiotics discovered in this era were predominantly produced by Streptomyces species. Streptomyces was reported to produce 70-80% of the all discovered antibiotics, which are mainly active as antibacterial and antifungal agents. However, there were some false classification of Steptomyces species, e.g., Streptomyces erythraeus, which was then amended to Saccharopolyspora erythraea. In this period, also, the discovery of other bioactivities such as antitumor, antiviral, and enzyme inhibitor had just commenced8,11.

In the 1970s-1990s, because of the cost of research had raised, the research had become laboriously. The novel compounds that were discovered in this period were primarily analogues of previously known metabolites. The area of research had become broad such as finding molecules for antitumor and agricultural antibiotics. In this period, problems of the emerging new pathogens and the growing of multi- resistant strains had become essential issues to be resolved. In the same time, it was also reported that many novel compounds produced from “rare actinomycetes” had been isolated and elucidated. This “rare actinomycetes” include the genera from actinomycetes beside Streptomyces such as Micromonospora, Actinomadura, Streptoverticillium, Actinoplanes, Nocardia, Saccharopolyspora and Streptosporangium8.

1 Introduction

From the 1990s until nowadays, the reports of the novel isolated compounds have been dramatically increased; however, these mostly belong to non-antibiotic molecules, analogous, and minor metabolites. The amount of novel chemical types had decreased. It is also recognized that there was an innovation gap in the period between 1962 and 2000, that means there were no primary classes of antibiotics introduced (Figure 2)8,12.

Chloramphenicol Quinolones Tetracyclines Streptogramins Oxazolidinones

Sulfa drugs Macrolides

Innovation gap

1940 1950 1960 2000 2010 Golden era

Mutilins β-Lactams Glycopeptides

Lipopeptides Aminoglycosides

Figure 2. Timeline of introduced antibiotics. Adapted from Fischbach and Walsh13.

On the other hand, the problems of multi-resistant strains, emerging new pathogens and re-emerging pathogens are increasing recently. The issues should be resolved; therefore, novel strategies are needed for discovering novel drugs. Some novel approaches for drug discovery have been carried out in recent times. These include the biodiversity-based method, modifying cultivation conditions, and genome mining8,14.

In the biodiversity based method, finding the novel species of bacteria is required in the effort of finding new antibiotics. There is a close relationship, based on the investigation during recent decades, between the discovering of new antibiotics and the description of novel bacteria species particularly within the Actinobacteria and gliding bacteria. It is suggested that the possibility of finding out novel compounds with unique structures are undoubtedly associated with the novel species. The method for isolation the novel species can be carried out by exploring the unexplored habitats such as desert, deep sea, and endosymbiotic environment. One of the underexplored countries, for Actinobacteria biodiversity, is Indonesia. Indonesia has an enormous territory encompassing 17,000 islands with diverse habitats and as one of the countries

1 Introduction having vast biodiversity in the world. Therefore, isolating Actinobacteria from Indonesian samples may increase the chance of either finding novel species or bioactive compounds. The other method which is still related to the biodiversity is by isolating and studying the bacteria which are not part of the taxa of eminent antibiotic producers14–16.

Some previous investigations suggested that the secondary metabolite profile of a bacterial species is possibly changed by modifying the culture conditions. By using whole-genome sequencing, it is finally understood that most fungi and bacteria have the capacity to generate more compounds than they usually produce in the standard cultivation condition. Changing the cultivation condition may be carried out by altering some parameters such as the concentration of phosphate in the medium, media composition, aeration, culture container, and addition of some enzyme inhibitors, solvents, and heavy metals. The other method is by using the co-culture method either with live or dead cells. By conducting such modifications, the probability to enhance the number of compounds produced by one strain is postulated become high. This method is known as OSMAC (One Strain-Many Compounds). The technique is suggested to stimulate the expression of the silent (cryptic) metabolic pathways in the microbial strains that can improve the variety of compounds they generate. However, this method is a random technique that is difficult to establish general standards for all strains14,17–19.

Genome mining is the method of retrieving information from genome sequences of species. This method can be conducted by detecting and analyzing the biosynthetic gene clusters of secondary metabolites and afterwards associating it to the corresponding chemical entities. Biosynthetic gene clusters are the main structures of the biosynthetic pathway of compounds at the genome level, which typically encode multidomain enzymes, such as polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), transporters, and some tailoring enzymes. The tailoring enzymes are some enzymes needed for a specific reaction such as halogenation, oxidation, glycosylation, and cyclisation. The determination of the biosynthetic gene cluster can be accomplished base on homology with notorious secondary metabolite gene clusters. This homology is obtained from the classification of previously recognized biosynthetic gene clusters based on the biosynthetic pathway, domain structure, conserved motifs, hidden Markov models, and chemical structure groups of encoded

1 Introduction compounds. The information derived from biosynthetic gene clusters can be employed to different methods such as guiding a more targeted drug discovery technique, peptide and glycogenomic approaches, and enabling the heterologous expression in the best expression host. However, it is still challenging to associate biosynthetic gene clusters to bioactivity14,15,20,21.

1.2 Actinobacteria The phylum Actinobacteria is one of the most prominent taxonomic and essential groups among the primary lineages within the Gram-positive bacteria group. They are mostly characterized by having high guanine and cytosine (G+C) content in their genomes Actinobacteria comprises soil dwellers (Streptomyces spp.), plant symbionts (Leifsonia spp.), nitrogen-fixing commensals (Frankia), and gastrointestinal tract inhabitants (Bifidobacterium spp.). However, small numbers of Actinobacteria are recognized as pathogens such as Mycobacterium spp. (e.g. Mycobacterium tuberculosis that causes tuberculosis (TB) in humans), Nocardia spp., Tropheryma spp., Corynebacterium spp., and Propionibacterium spp. Most of Actinobacteria are aerobic, heterotrophic, and can be found in both terrestrial and aquatic ecosystems (including marine habitats)22,23.

They generate about two-thirds of all clinically used antibiotics from natural products. Many Actinobacteria produce a mycelium like fungi. Because of this, they are considered as the transitional organisms between fungi and bacteria. The mycelium formed Actinobacteria reproduce by sporulation and are called actinomycetes, which originated from the Greek words for ray (aktis or aktin) and fungi (mukēs). The difference between actinomycetes and fungi is that the former have no nucleus, contain peptidoglycan in their cell wall, and are susceptible to antibacterial agents. Most of them are saprophytic and soil-dwelling bacteria that spend most of their life cycles as semi-dormant spores, particularly in the condition when their nutrients are scarce. They mainly grow better at a pH 6-9, with the most optimal growth around neutrality and are mostly mesophilic, which means that the temperature for their optimal growth is between 25-30°C22.

The phylum Actinobacteria has six classes. These include Acidimicrobiia, Actinobacteria, Coriobacteriia, Nitriliruptoria, and Thermoleophilia. The class Actinobacteria encompasses 29 orders and 62 families, as shown in Table 1. From 62 families in the phylum Actinobacteria, around 74% of them belong to the class

1 Introduction

Actinobacteria (https://www.ncbi.nlm.nih.gov/taxonomy). Delineation of species in Actinobacteria is conducted by phenotypic and genotypic analysis. The primary phenotypic characteristics employed to delineate the taxonomy of Actinobacteria are based on the microscopic morphology and chemotaxonomy. For genotypic analysis, the taxonomic study of Actinobacteria can be carried out based on the 16S rRNA gene analysis, DNA-DNA hybridization, multilocus sequence analysis (MLSA), and genome sequencing22,24.

Table 1. Current taxonomic outline for the phylum Actinobacteria. The data are based from taxonomy database in NCBI website (https://www.ncbi.nlm.nih.gov/taxonomy)

Phylum Class Order Family Genus Actinobacteria Acidimicrobiia, Acidimicrobiales 4 Families 10 genera Actinobacteria, Acidothermales 1 Family 1 genera Actinomycetales 1 Family 11 genera Actinopolysporales 1 Family 2 genera Bifidobacteriales 1 Family 10 genera Catenulisporales 2 Families 3 genera Corynebacteriales 7 Families 20 genera Cryptosporangiales 1 Family 2 genera Frankiales 2 Families 4 genera Geodermatophilales 1 Family 5 genera Glycomycetales 1 Family 5 genera Jiangellales 1 Family 3 genera Kineosporiales 1 Family 6 genera Micrococcales 16 161 Micromonosporales Families genera Nakamurellales 1 Family 33 genera Propionibacteriales 1 Family 1 genera Pseudonocardiales 2 Families 33 genera Sporichthyales 1 Family 35 genera Streptomycetales 1 Family 3 genera Streptosporangiales 1 Family 6 genera 3 Families 35 genera Coriobacteriia, Coriobacteriales 2 Families 10 genera Eggerthellales 1 Family 15 genera Nitriliruptoria, Egibacterales 1 Family 1 genera Egicoccales 1 Family 1 genera Euzebyales 1 Family 1 genera Nitriliruptorales 1 Family 1 genera Thermoleophilia Solirubrobacterales 4 Families 4 genera Thermoleophilales 1 Family 1 genera

1 Introduction

1.2.1 Streptomyces Bacteria of the genus Streptomyces can produce a secondary metabolite named geosmin. This compound does not have any antibiotic activity; however, it gives the soil its distinctive smell. They are perfectly adapted to survive in the soil where they grow by forming substrate mycelium that can help them to get nutrients. They secrete various enzymes that can digest insoluble organic polymers and numerous secondary metabolites that may be suggested not only as chemical weapons for killing other soil organisms but also as signalling molecules for modulating the metabolic process in target organisms. They also have antibiotic resistance genes to protect themselves from their own produced antibiotics. These resistance genes, however, can be transferred to other bacteria via horizontal gene transfer. The life cycle of Streptomyces starts with spore germination, forming vegetative hyphae, and continues to create substrate mycelium. The exponential growth of the vegetative hyphae is accomplished through a combination of tip extension and branching. The cell division during vegetative growth creates cross-walls that separate the hyphae into connected compartments. In response to unfavourable conditions such as nutritional deficiency and other stress signals, the vegetative mycelium differentiates to form reproductive aerial hyphae, which undergo cellular division to produce spores. Most of the antibiotics are formed in this differentiation moment; however, many industrial processes for secondary metabolite production from Streptomyces are accomplished with liquid cultures. Under these conditions, Streptomyces strains usually do not sporulate22,25,26. The morphology of Streptomyces species can be seen in Figure 3.

Streptomyces strains can produce various antibiotics with different structures and functions such as inhibition of DNA replication and the synthesis of RNA, cell wall, and protein. Some examples of antibiotics produced by Streptomyces species are streptomycin from S. griceus, cephalosporin from S. clavuligerus, chloramphenicol from S. venezuelae, daptomycin from S. roseosporus, and novobiocin from S. niveus. Streptomycin and chloramphenicol are the inhibitors of bacterial protein synthesis. Streptomycin binds to the small 16S rRNA of the 30S ribosomal subunit, while chloramphenicol binds to 23S rRNA of the 50S ribosomal subunit. Cephalosporin was firstly isolated from fungus Acremonium chrysogenum and then it was known that Streptomyces also can produce the compound. It disrupt the synthesis of the bacterial

1 Introduction cell wall, while daptomycin can break up the cell membrane. As for novobiocin, it has another mechanism of antibacterial activity by binding to DNA gyrase27,28. Some other antibiotics produced by Streptomyces species are shown in Table 2.

Figure 3. Streptomyces sp. strain C196921 in agar medium

Table 2. Some antibiotics produced by Streptomyces species

Antibiotic Class of molecule Function Producer Reference Arylomycin Lipopeptides Inhibition of Streptomyces sp. 29,30 type I signal Tu 6075 peptidase Capreomycin Peptides Inhibition of S. vinaceus 31 protein S. capreolus synthesis Cephalosporins β-Lactams Inhibition of S. clavuligerus 32,33 cell wall synthesis Chloramphenicol Chloramphenicols Inhibition of S. venezuelae 34,35 protein synthesis Cycloserine Analog of D- Inhibition of S. garyphalus 36,37 alanine cell wall synthesis Daptomycin Lipopeptides Destruction of S. roseosporus 38 the membrane potential

1 Introduction

Fosfomycin Fosfomycin Inhibition of S. fradiae 39 cell wall synthesis Kanamycin Aminoglycosides Inhibition of S. kanamyceticus 40 protein synthesis Lincomycin Lincosamides Inhibition of S. lincolnensis 41 protein synthesis Neomycin Aminoglycosides Inhibition of S. fradiae 42,43 protein synthesis Novobiocin Aminocoumarins Inhibition of S. niveus 44,45 DNA gyrase Oleandomycin Macrolides Inhibition of S. antibioticus 46,47 protein synthesis Platensimycin Platensimycin Inhibition of S. platensis 48 fatty acid production Pristinamycin Streptogramins Inhibition of S. 49 protein pristinaespiralis synthesis Ribostamycin Aminoglycosides Inhibition of S. ribosidificus 50 protein synthesis Spiramycin Macrolides Inhibition of S. ambofaciens 51 protein synthesis Streptomycin Aminoglycosides Inhibition of S. griseus 52 protein synthesis Tetracycline Tetracyclines Inhibition of S. aureofaciens 53,54 protein synthesis Viomycin Peptides Inhibition of S. vinaceus, 31 protein S. capreolus synthesis Virginiamycin Streptogramins Inhibition of S. virginiae 55 protein synthesis

1.2.2 Non-Streptomyces Actinobacteria In contrast to the member of the genus Streptomyces that produce around 74% of antibiotics from Actinobacteria sources, non-Streptomyces Actinobacteria contribute

1 Introduction only about 26% of the antibiotics of their origin. This non-Streptomyces Actinobacteria are called rare actinomycetes, which comprise more than 200 genera e.g., Actinomadura, Actinoplanes, Amycolatopsis, Actinokineospora, Acrocarpospora, Actinosynnema, Catenuloplanes, Cryptosporangium, Dactylosporangium, Kibdelosporangium, Kineosporia, Kutzneria, Microbiospora, Micromonospora, Microtetraspora, Nocardia, Nonomuraea, Planomonospora, Planobispora, Pseudonocardia, Saccharomonospora, Saccharopolyspora, Saccharothrix, Streptosporangium, Streptoverticillium, Spirilliplanes, Thermomonospora, Thermobifida, and Virgosporangium8,56. The morphology of some non-Streptomyces can be seen in Figure 4.

Rare actinomycetes are frequently referred to the strains of Actinobacteria whose isolation rate is considerably less than that of the Streptomyces strains isolated by classical approaches. In comparison with Streptomyces, they have not been extensively investigated in previous years and might become new sources of novel secondary metabolites. These bacteria are extensively distributed both in terrestrial and aquatic ecosystems. Their distribution is influenced by some environmental elements such as soil type, pH, humus content, and the characteristics of the humic acid content of the soil. While non-Streptomyces Actinobacteria may increase the probability of discovering novel compounds, their genetics and physiology are not well understood56.

Amycolatopsis sp. Pseudonocardia sp. Microbispora sp. strain 196526CR strain 5931 strain 190401

Figure 4. Some non-Streptomyces strains in agar medium

The prolific group of non-Streptomyces Actinobacteria is represented by Actinoplanes strains, which have significantly been isolated by utilizing the properties of their

1 Introduction spores that are mobile and have chemotactic movement. These groups of bacteria produce more than 120 antibiotics such as teicoplanin, ramoplanin, purpuromycin, lipiarmycin, and actagardine. Another group of non-Streptomyces Actinobacteria isolated on an enormous scale over the last few years belong to the genus Micromonospora. This genus is regarded as the second biggest group of culturable Actinobacteria in soil and can be isolated by using selection medium containing antibiotics such as gentamicin and novobiocin or with the pre-treatment by using toxic agents such as phenol and chlorhexidine gluconate solutions because their spores are resistant to these chemicals. Micromonospora strains produce some important antibiotics such as gentamicin, sisomicin, fortimicin, mycinamicins, rosamicins, and everninomycin57. Some other antibiotics produced by non-Streptomyces species are listed in Table 3.

1.3 Polyphasic taxonomy of the class Actinobacteria Taxonomy, which is the synonym of systematics or biosystematics, is essential in studying organisms. It comprises three main parts, i.e., classification, nomenclature, and identification. Classification can be carried out by organizing organisms based on similarity into taxonomic groups. The term nomenclature includes giving the label of the units determined in classification. Identification means the process of deciding whether an organism is a member of one of the units determined in classification and labelled in nomenclature. Taxonomic information helps scientists to figure out the biodiversity and correlation among organisms from distinct ecological systems. Taxonomy in prokaryotes has an indispensable function in supporting the accurate identification of microbial strains from difference varieties58,59.

Table 3. Some antibiotics produced by non-Streptomyces species

Antibiotic Class of molecule Function Producer Refe- rence Abyssomycin Spirotetronates Inhibition of Verrucosispora sp. 60 folate AB-18-032 biosynthesis Actagardine Polycyclic Inhibition of Actinoplanes 61 peptides cell wall garbadinensis synthesis Borrelidin Macrolides Inhibition of Nocardiopsis sp. 62,63 protein HYJ128 synthesis

1 Introduction

Branimycin Macrolides Inhibition of Pseudonocardia 64,65 DNA synthesis carboxydivorans M‑227 Cationomycin Polyethers Modification Actinomadura 66 of cell azurea membrane Everninomycin Oligosaccharides Inhibition of Micromonospora 67,68 protein carbonacea synthesis Fortimicin Aminoglycosides Inhibition of Micromonospora 69,70 protein olivoastrospora synthesis Gentamicin Aminoglycosides Inhibition of Micromonospora 71 protein purpurea synthesis Kibdelomycin Kibdelomycin Inhibition of Kibdelosporangium 72 DNA synthesis sp. MA7385 Kijimicin Polyethers Modification Actinomadura sp. 73 of cell MI215- NF3 membrane Lipiarmycin Macrolides Inhibition of Actinoplanes 74,75 RNA synthesis deccanensis Madurahydroxylactone Benzo[a]naphtha- Inhibition of Nonomuria rubra 76,77 cenequinones cell-division protein FtsZ Mycinamicin Macrolides Inhibition of Micromonospora 78,79 protein griseorubida synthesis Nargenicin Macrolides Inhibition of Nocardia devorans, 80,81 DNA synthesis Nocardia sp. CS682 Purpuromycin Naphthoquinones Inhibition of Actinoplanes 82,83 protein ianthinogenes synthesis Ramoplanin Lipodepsipeptides Inhibition of Actinoplanes 84,85 cell wall ramoplaninifer synthesis Rifamycin Ansamycins Inhibition of Amycolatopsis 86 RNA synthesis mediterranei Rosamicin Macrolides Inhibition of Micromonospora 87,88 protein rosaria synthesis Saccharomicin Oligosaccharides Disruption of Saccharothrix 89 cell membrane espanaensis

1 Introduction

Sisomicin Aminoglycosides Inhibition of Micromonospora 90,91 protein inyoensis synthesis Teicoplanin Glycopeptides Inhibition of Actinoplanes 92 cell wall teichomyceticus synthesis Vancomycin Glycopeptides Inhibition of Amycolatopsis 93 cell wall orientalis synthesis

Bacterial taxonomy commenced in the late 19th century when the classification was only based on simple phenotypic markers such as morphology, growth requirements or pathogenicity. Subsequently, the other properties, i.e., physiological and biochemical, were added for this purpose. In the period of 1960s-1980s, chemotaxonomy, numerical taxonomy and DNA–DNA hybridization techniques were applied in studying taxonomy. Later, in the 1980s, the emergence of DNA amplification and sequencing techniques, especially of the 16S rRNA gene, created significant progress in bacterial classification. Since the mid-1990s, whole-genome sequencing cretaed an innovation by providing an approach to obtain the complete genetic information of a single strain59.

In order to establish reliable taxonomy of bacteria, integration of phenotypic, genotypic, and phylogenetic information of the strains is needed, and this is called polyphasic taxonomy. Phenotypic information is derived from main physical characteristics such as morphology, staining properties, ultrastructure, chemotaxonomic markers, physiological properties, biochemical features, protein composition, and pathogenesis. Meanwhile, genotypic data are obtained from the nucleic acids (DNA and RNA) in the cell such as analysis of DNA–DNA hybridization (DDH), DNA G+C content and 16S rRNA gene sequencing58,59,94. Some other methods recently used for genomic taxonomy study include ribotyping and whole- genome analysis95,96. Phylogeny depicts biological entities that are connected through common descent. It can be species, genus or higher-level taxonomic groups. A phylogenetic tree is used for understanding not only the relationships among taxa (or sequences) but also their hypothetical related predecessors. Currently, most phylogenetic trees are constructed from molecular data such as DNA or protein sequences.

1 Introduction

As mentioned above, the class of Actinobacteria is an essential group in both health and economy that consists of 29 orders and 62 families. In the effort of discovering drugs from Actinobacteria sources, polyphasic taxonomy is needed either for establishing reliable identification of antibiotics producing strains or as a dereplication tool to avoid redundancy in the selection of Actinobacteria species97. Microscopic morphology and chemotaxonomy are the primary characteristics employed to delineate the taxonomy of Actinobacteria at the genus and species levels. However, with the recent development in molecular analysis, some species that were inadequately positioned in particular taxonomic groups have recently been reclassified using molecular analyses22.

1.3.1 Phenotypic analysis Various morphologies can be found in Actinobacteria, which include coccoid (Micrococcus), rod-coccoid (Arthrobacter), irregular rods (Propionibacterium), fragmenting hyphal forms (Nocardia), and permanent and highly differentiated branched mycelia (Streptomyces, Kitasatopora and Frankia). The members of genus Rhodococcus form elongated filaments without producing a real mycelium. The members of genus Corynebacterium produce no mycelia. There is also a group that can break up its hyphae into flagellated motile elements (Oerskovia). Some members of Mycobacterium and Rhodococcus mostly do not form aerial hyphae22,98,99.

Spore is one of the important markers in the taxonomy of Actinobacteria, although some Actinobacteria produce no spores, e.g., the members of Bifidobacterium, Micrococcus, and Propionibacterium. Spores may be produced on the substrate only (Micromonospora) or both on the substrate and the aerial mycelium (Streptomyces) either as single cells or in chains with several of length. In some groups, spores may be sheltered in specialized vesicles called sporangia (Actinoplanes, Planomonospora, Planobispora, Dactylosporangium, and Streptosporangium) and equipped with flagella for the motility of spores (Actinoplanes and Actinosynnema)22,100,101.

Some Actinobacteria can produce various pigments, including melanin based on the medium that is used and the age of the culture. These may be red, yellow, orange, pink, brown, greenish-brown, blue, or black. Melanins are polymers with various structures that usually appear black or brown and are derived from oxidative polymerization of phenolic and indolic molecules. They are not necessary for the growth and development, but they can help the host for survival and competition to

1 Introduction other organisms. They can be used in taxonomic studies and have a similar character to soil humic substances22. Investigation of pigments including melanin produced by Actinobacteria and their features of the morphology such as substrate mycelia, aerial mycelia, and spores can be conducted by utilizing International Streptomyces Project (ISP) medium102. The observation of spores formed by Actinobacteria is conducted employing a scanning electron microscope (SEM)103.

As mentioned above, physiological and biochemical properties are parts of phenotypic criteria in the taxonomy study. Physiological data useful for Actinobacteria classification determinations include growth temperature, pH value, salt tolerance, growth on sole carbon sources, antibiotic resistance and oxygen requirement whereas biochemical features of interest are carried out by enzymatic activity test100,102,104–107.

In the classification of Actinobacteria, the analysis of chemical components in the cell such as the analysis of cell-wall amino acids, lipids, proteins, menaquinones, muramic acid types, and sugars, is required22. Some genera contain 2, 6-diaminopimelic acid (DAP) in their cell-wall structure, which has isomers, i.e., LL-DAP and meso- DAP. Streptomyces, Sporichthya, and Intrasporangium are the example of genera that have only LL-DAP. For meso-DAP, it can be found in the members of Mycobacterium, Nocardia, and Pseudonocardia. Hydroxy-diaminopimelic acid (OH-DAP), which is the derivative of DAP, is detected in Micromonospora strains. For whole-cell sugar patterns, Streptomyces species have no characteristic sugar pattern. Mycobacterium, Nocardia, and Pseudonocardia species have galactose and arabinose as their whole sugar pattern. Microbispora and Streptosorangium strains contain madurose, whereas the members of Actinoplanes, Dactylosporangium, and Micromonospora, possess xylose and arabinose108.

Lipids, such as fatty acids, phospholipids, and menaquinones, have been used for chemotaxonomy of Actinobacteria. Fatty acids iso or anteiso C15:0, C15:0, or C17:0 have been found as the predominant fatty acid in Rothia, Actinoplanes, Nocardiopsis, Amycolatopsis, and Streptomyces species106,109–112. Diphosphatidyl glycerol, phosphatidyl inositol mannosides and phosphatidyl inositol are mostly found in Actinobacteria species. Many Actinobacteria possess phosphatidyl ethanolamine except the members of Actinomadura, Corynebacterium, Microtetraspora, and Nocardioides. Phosphatidylcholine can be found in Actinomadura, Nocardia and Pseudonocardia strains113. As for menaquinones, some Corynebacterium species

1 Introduction contain menaquinone MK-8(H2) as the predominant menaquinone. MK-8(H4) is the primary menaquinone system in Nocardia members. Many Mycobacterium strains possess menaquinone MK-9(H2) as the primary menaquinone system. The menaquinone compounds in some Streptomyces are dominated with MK-9(H6), whereas in Actinomyces species, MK-10(H4) is the main menaquinone they have114.

Some taxonomy studies in Actinobacteria were reported analyzing ribosomal protein using matrix-assisted laser-desorption/ionization time-of-flight spectrometry (MALDI-TOF MS)109,115,116. Ribosomal proteins have also been used for taxonomy study of bacteria since their peaks in MALDI-TOF MS are dominant, they are rather conservative molecules in the context of evolution, and their spectra are also specific of a particular strain and undergo only a little change with different culture conditions117.

1.3.2 Genomic and phylogenetic analysis The genomic DNA G+C content is the ratio of guanine and cytosine within the total number of nucleotides in the genome. It is one of the taxonomic markers and can be employed for discriminating phenotypically similar microorganisms. It is also the primary characteristic of cellular DNA and is related with the amino acid composition of proteins, codon usage in mRNA, auxotrophy for specific bases, and other features of overall biological importance118,119. Class of Actinobacteria is encompassed mostly of Gram-positive bacteria with a high G+C content (>55 mol% in genomic DNA). Some members of Corynebacterium, however, have G+C content lower than 55% and even Gardnerella strains have G+C content which is less than 45%23,120.

Ribosomal RNA has been used in the taxonomic study due to its availability in all self- replicating systems, ease of isolation, and its sequence changes very slowly. 16S ribosomal RNA (16S rRNA) is one of the ribosomal RNAs in bacteria. 16S rRNA gene is used as the backbone for the classification of bacteria because it is ubiquitous, functionally stable, highly conserved and poorly subject to horizontal gene transfer (HGT). In the recent decade, comparative analysis of the 16S rRNA sequence, which has a size around 1,500 nucleotides, has been employed to study prokaryote phylogeny. In the 16S rRNA gene sequence analysis, the cut-off of identity values for determining novel genus is 95%, whereas it is 98.7% for novel species (previously it was 97%), when the 16S rRNA gene sequence of the tested bacterial strains are compared with their phylogenetically closest neighbours with validly published

1 Introduction names59,121–123. In the case of Streptomyces species, previous studies found that there are some novel Streptomyces species, which have the similarity value of 16S rRNA gene sequence comparison 99.1-99.9%124–128.

Ribotyping is a genetic fingerprinting method, which uses digested genomic DNA, separating it by gel electrophoresis, and hybridizing it with ribosomal DNA (rDNA) probe by Southern blotting technique. The restriction enzymes that are used can be EcoRI, PstI, PvuII, BamHI, ClaI and HindIII. The complete ribosomal operon from Escherichia coli is employed as the rDNA probe. The resulted pattern is a genetic fingerprint, which is useful to discriminate bacterial strains. Many bacterial species can be delineated with Ribotyping method, including Actinobacteria95,129.

DNA–DNA hybridization is a commonly used technique to evaluate the genetic relationship between bacterial strains and is still known as the ‘gold standard’ principle for species delineation of prokaryotes. The tested bacterial strains are indicated as distinct species when their DDH values are ≤ 70%. DDH, however, has some shortcomings such as the cut-off values are not pertinent to all prokaryote genera, determining DDH values needs special facilities therefore the method is not available in many laboratories, and it is a laborious and expensive method that has shortage of reproducibility and is challenging to create a comparative reference database progressively from DDH data59.

Multi-locus sequence analysis has been used for delineation some bacterial species in several genera, including members of Actinobacteria such as Mycobacterium, Streptomyces and Kitasatospora. By using the concatenation of partial gene sequences from five house-keeping genes, the closely related species of Streptomyces can be discriminated. These five house-keeping genes are atpD (ATP synthase F1, beta subunit), gyrB (DNA gyrase B subunit), recA (recombinase A), rpoB (RNA polymerase, beta subunit) and trpB (tryptophan synthase, beta subunit). This method is reproducible, can allow creating cumulative databases, and is comparable to DDH. The five-gene MLSA distance of 0.007 corresponds to a DDH value of 70%, which means that this value could be employed as the species cut-off for the whole Streptomyces genus. The sequences derived from the concatenated genes can also be used for generating phylogenetic tree 24,130,131.

1 Introduction

Whole-genome data has also been used in bacterial taxonomy (including Actinobacteria), i.e., by aligning and comparing whole-genome sequences between two genome sequences from the closely related strains. The term overall genome relatedness index (OGRI) is coined to represent any measurements showing how identical two tested genome sequences are. The most commonly used algorithm for taxonomic investigations is average nucleotide identity (ANI). When comparing to DDH, 95-96% ANI value is equivalent to 70% DDH. It means ANI value can be employed to delineate novel species in bacteria96,132,133.

1.4 Dereplication and isolation of natural products from Actinobacteria Discovering of bioactive molecules from Actinobacteria sources nevertheless requires substantial investments in technical equipment, time and human resources. Rapid identification of known substances, which is called dereplication, is one of the important strategies to focus the efforts on the finding of novel bioactive compounds. The term of dereplication nowadays, however, is a not so univocal and has developed in the past few years in different ways. There are many dereplication methods developed to enhance the success of natural product discovering programs134,135.

Dereplication approach includes some methods, e.g., biological screening processes, LC-MS (liquid chromatography-mass spectrometry) techniques with the combination of MS libraries and databases, NMR (nuclear magnetic resonance) spectroscopy, and HTS (high throughput screening) technology. Taxonomic identification using 16S rRNA gene sequence has been used since the early of the 2000s as a dereplication tool. Bioactivity-guided assays with combination LC-MS have also been employed for dereplication approach97.

Hubert et al.135 reviewed dereplication strategies in natural product research. They classified dereplication approaches into five categories, i.e., DEREP1, DEREP2, DEREP3, DEREP4, and DEREP5. Identification of the major compounds in a single extract is the activity in DEREP1. Systematically biological assay is included in DEREP2 (acceleration of activity-guided fractionation). DEREP3 is chemical profiling of crude extract collections. Chemical profiling of target compounds is conducted in DEREP4. The last, DEREP5 is the dereplication with taxonomic identification of microbial strains. Dereplication of natural products from Actinobacteria sources, therefore, may be conducted by using 16S rDNA analysis and with the combination of LC-MS-bioassay method.

1 Introduction

Isolation of natural products from Actinobacteria may be carried out following the bioassay-guided isolation strategies that connect information on the chemical profiles of extracts and the active fractions. Extraction procedures employ organic solvents of different polarity, water and the mixtures of them or using solid-phase extraction (SPE) method136. Bioactivity assays can be done with antibacterial, antifungal, anticancer, or antiviral activity, e.g., antihepatitis virus, which is described briefly below. The antimicrobial test may be conducted either by using the zone inhibition test137 or serial dilution test with 96-well plate138. The further isolation process needs preparative thin layer chromatography (TLC) or column chromatographic methods. Finally, for the structure elucidation of the isolated compound, high resolution mass spectrometry (HR-MS) and NMR spectroscopy is employed136,139. HR-MS can provide accurate mass measurements that is needed for determination of chemical formula resulting high confidence in the structural elucidation139. As for NMR, it is used for determining the molecular structure of the compound140.

1.5 Hepatitis Hepatitis is a disease identified by inflammation of the liver tissue and is caused mostly by viruses. There are five types of hepatitis viruses, i.e., type A, B, C, D, and E. Hepatitis A and E are usually caused by consuming contaminated food or water, while hepatitis B happens frequently through contact with infected bodily fluids like blood or semen. Hepatitis C virus is a blood-borne virus that is mostly transmitted by the usage of sharing needles from person to person. As for hepatitis D virus, it is spread with the infectious blood and the disease occurs only among the patients who have been infected by hepatitis B virus. Hepatitis B, C, and D plague more than half a billion people throughout the world and cause more than a million deaths annually141.

Hepatitis A virus is a nonenveloped RNA with 7.5-kb genome size and belongs to the genus Heparnavirus of the Picornaviridae family142. Hepatitis B virus is a DNA virus and a member of the genus Orthohepadnavirus in the Hepadnavidae family143. Hepatitis C virus (HCV) is a member of the genus Hepacivirus in the family Flaviviridae. It is a positive single-stranded RNA virus with circa 9.6 kb size. It mainly propagated in the hepatocyte cytoplasm and mostly causes acute or chronic hepatitis C (CHC)144. Hepatitis D virus is an RNA virus and belongs to Deltavirus genus in the Deltaviridae family. It is a satellite virus of HBV because its incapability of infection in the absence of Hepatitis B virus145. Hepatitis E virus (HEV) is a single

1 Introduction stranded and a positive RNA virus. It belongs to the Orthohepevirus genus in the Hepeviridae family. Its genome size is 7.2 kb and it is the primary cause of global enterically transmitted hepatitis146.

1.6 Previous work In the course of Actinobacteria investigation from the neglected habitat sources, strain ASO4wet was isolated from the sponge in a deep sea. In December 2014, the Team of Prof. Dr. Peter Schupp (University of Oldenburg) took samples from marine sediment from 1092 m depth in the North Atlantic Ocean during an expedition with a new marine research vessel called Sonne. Study of 16S rRNA gene sequences revealed that strain ASO4wet is a member of the genus Streptomyces and exhibited the closest similarities to S. karpasiensis (98,94%), S. glycovorans (98%) and S. abyssalis (98%). DNA-DNA hybridization (DDH) analyses between strain ASO4wet and its closest related type strains indicated that strain ASO4wet is a distinct species from its compared closest species. Some studies have been conducted regarding to the novel description of the novel species for strain ASO4wet, including morphology and biochemical comparison, DDH, ribotyping, and MALDI-TOF analysis. From these studies, it is known that strain ASO4wet is different based on the morphologic, biochemical, and genomic characteristic from its close neighbours. However, chemotaxonomy study such as fatty acid, phospholipid, cell-wall amino acid, and whole-cell sugar analysis, of isolate ASO4wet has not yet been conducted and therefore, it needs to be carried out in order to fulfil the requirement of polyphasic taxonomy of strain ASO4wet147.

1.7 Aim of study The objectives of the research are:

1) Isolation of Actinobacteria from Indonesia 2) Cultivation and induction of secondary metabolite production (by using different media) 3) Extraction and screening of bioactivity, including antimicrobial and antiviral activity 4) Analysis, isolation and structure elucidation of metabolites produced by isolated Actinobacteria 5) Taxonomic study of selected Actinobacteria strains including strain ASO4wet

2 Materials and Methods

2 Materials and Methods 2.1 Materials 2.1.1 Media All of the media were sterilized for 20 min at 121°C.

Table 4. List of media used in this study.

Medium Composition Amount pH or Manufacturer ISP medium (International Streptomyces Project)

ISP2/ Yeast malt agar Malt extract 10.0 g/l 7 Maintenance and taxonomy Yeast extract 4.0 g/l Glucose 4.0 g/l Agar 15.0 g/l Deionized Water 1000 ml ISP3/ Oat meal agar Oatmeal (Quaker white oats) 20.0 g/l 7.2 Maintenance and taxonomy Agar 18.0 g/l Deionized Water 1000 ml Trace salt solution 1 ml

Trace salt solution ISP3 FeSO4 x 7H2O 0.1 g

MnCl2 x 4H2O 0.1 g

ZnSO4 x 7H2O 0.1 g Deionized water 100ml ISP 4 Soluble starch 10.0 g/l 7.3

Maintenance and taxonomy (NH4)2SO4 2.0 g/l

K2HPO4 1.0 g/l

MgSO4 x 7H2O 1.0 g/l NaCl 1.0 g/l

CaCO3 2.0 g/l Agar 20.0 g/l Deionized Water 1000 ml ISP5 L-Asparagine 1.0 g/l 7.2 Maintenance and taxonomy Glycerol 10.0 g/l

K2HPO4 1 g/l Trace salt solution 1ml/l Agar 20 g/l Deionized Water 1000 ml Trace salt solution ISP5 1,0 g FeSO4 x 7 H2O 1.0 g 1,0 g MnCl2 x 4 H2O 1.0 g 1,0 g ZnSO4 x 7 H2O 1.0 g

2 Materials and Methods

Deionized water 100 ml ISP6 / Peptone Iron Agar Peptone 15.0 g/l 7.2 Production of melanoid Proteose Peptone 5.0 g/l pigment Ferric ammonium citrate 0.5 g/l Sodium glycerophosphate 1.0 g/l Sodium thiosulfate-5-hydrate 0.126 g/l Yeast extract 1.0 g/l Agar 20 g/l Deionized Water 1000 ml ISP7/ Oat meal agar Glycerol 15 g/l 7.3 Production of melanoid L-Tyrosine 0.5 g/l pigment L-Asparagine 1.0 g/l

K2HPO4 0.5 g/l NaCl 0.5 g/l

FeSO4 x 7 H2O 0.01 g/l Trace Salt solution (5343) 1.0 ml/l Agar 20.0 g/l Deionized water 1000 ml GYM/ Streptomycetes Glucose 4.0 g/l 7.2 Medium Yeast extract 4.0 g/l Maintenance and Malt extract 10.0 g/l revitalization CaCO3 2.0 g/l Agar 12 g/l Deionized Water 1000 ml GYM + ASW GYM medium 1000 ml 7.2 (Artificial Sea Water) Coral reef salt “Coral Ocean”, ATI 39 g Synthetically Suter Glycerol 15.0 g/l 7.2 Medium Tyrosine 1.0 g/l Production of melanoid L-arginine 5.0 g/l pigment L-glutamic acid 5.0 g/l

L-methionine 0.3 g/l L-isoleucine 0.3 g/l K2HPO4 0.5 g/l MgSO4 x 7 H2O 0.2 g/l Trace element solution 2 (5341) 1.0 ml/l Agar 20 g/l -Control medium is prepared without tyrosine

Basal medium for (NH4)2SO4 2.64 g/l 7.3 carbohydrate utilization KH2PO4 2.38 g/l

K2HPO4 4.31 g/l

MgSO4 x 7 H2O 1.0 g/l Agar 15.0 g/l

2 Materials and Methods

Trace element solution 3 (5342) 1.0 ml/l Deionized Water 1000 ml

5341 CuSO4 x 5 H2O 10.0 g/l

Trace element solution 2 CaCl2 x 2 H2O 10 g/l

FeSO4 x 7 H2O 10 g/l

ZnSO4 x 7 H2O 10 g/l

MnSO4 x 7 H2O 40 g/l Deionized water 1000 ml

5342 CuSO4 x 5 H2O 0.64 g/l

Trace element solution 3 FeSO4 x 7 H2O 0.11 g/l

ZnSO4 x 7 H2O 0.15 g/l

MnCl2 x 4 H2O 0.79 g/l Deionized water 1000 ml

5343 FeSO4 x 7 H2O 1 g/l

Trace element solution 4 ZnSO4 x 7 H2O 1 g/l

MnCl2 x 4 H2O 1 g/l Deionized water 1000 ml Sodium chlorite tolerance Casein peptone 10.0 g/l 7.0 Yeast extract 5.0 g/l Agar 20 g/l Deionized Water 1000 ml 5006 Sucrose 3.0 g/l 7.2 sterility control Dextrin 15.0 g/l Meat extract 1.0 g/l Yeast extract 2.0 g/l Tryptone soy broth 5.0 g/l NaCl 0.5 g/l

K2HPO4 0.5 g/l

MgSO4 x 7 H2O 0.5 g/l

FeSO4 x 7 H2O 0.01 g/l Agar 20 g/l Deionized Water 1000 ml 5336 Soluble starch 10.0 g/l 7.3 Actinobacteria isolation Casein (Pepton Typ M) 1.0 g/l medium K2HPO4 0.5 g/l

MgSO4 x 7 H2O 5.0 g/l Agar 20.0 g/l Deionized water 1000 5336 + Cyclo + NA Sterilized 5336 medium 1000 ml 7.3 Actinobacteria isolation Cyclohexamide solution (50 mg/ml 2 ml medium in methanol) Nalidixic acid solution (25 mg/ml 1 ml in 0.2 M NaOH)

2 Materials and Methods

Medium 5254 Glucose 15.0 g/l 7.0 Metabolite production Soymeal 15.0 g/l Corn steep liquor 5.0 g/l

CaCO3 2.0 g/l NaCl 5.0 g/l Deionized water 1000 ml Medium 5294 Starch (soluble) 10.0 g/l 7.2 Metabolite production Yeast extract 2 g/l Glucose 10 g/l Glycerol 10 g/l Corn steep liquor 2.5 g/l Peptone (Marcor S) 2.0 g/l NaCl 1 g/l CaCO3 3.0 g/l Deionized water 1000 ml Medium 5294HG-S Yeast extract 2 g/l 7.2 Metabolite production Glucose 20 g/l Glycerol 10 g/l Corn steep liquor 2.5 g/l Peptone (Marcor S) 2.0 g/l NaCl 1.0 g/l CaCO3 3.0 g/l Deionized water 1000 ml 5254 + ASW 5254 medium 1000 ml 7.0 (Artificial Sea Water) Coral reef salt “Coral Ocean”, ATI 39 g 5294 + ASW 5294 medium 1000 ml 7.2 (Artificial Sea Water) Coral reef salt “Coral Ocean”, ATI 39 g SYP medium Starch (soluble) 10 g 7.0 Yeast extract 4 g Peptone (Marcor S) 2 g Deionized water 1000 ml Middelbrock Broth medium Becton, Dickinson and Company, France Müller-Hinton Bouillon Carl Roth GmbH + Co.KG, (MHB) Germany Myc medium Phytone peptone 0.1 g 7.0 Glucose 0.1 g HEPES 11.9 g Deionized water 1000 Trypticase soy broth Becton, Dickinson and Company, France Tryptone soy broth Oxoid

2 Materials and Methods

2.1.2 Chemicals Table 5. List of chemicals used in this study.

Chemical Manufacturer 1 kb DNA ladder BioLabs Acetic acid Carl Roth Acetone J.T. Baker Acetonitrile J.T. Baker Agarose Gibco BRL Ammonium acetate Carl Roth

Ammonium iron(III)citrate ((NH4)5Fe(C6H4O7)2) Carl Roth

Ammonium Sulfate (NH4)2SO4 Merck Aniline phthalate spray solution for TLC Carl Roth Anisaldehyde solution spray reagent Sigma-Aldrich Arabinose Merck Bacto Agar® Becton Dickinson

Calcium carbonate (CaCO3) Panreac Appli Chem Casein (Pepton Typ M) Marcor Cellulose Serva, Heidelberg

Copper(II) sulfate (CuSO4 x 5 H2O) Merck Corn steep liquor Schering Dimethylsulfoxid (DMSO) Carl Roth Dulbecco´s modified Eagle´s medium (DMEM) Bio Whittaker, Walkersville, MD Cyclohexamide Serva Ethanol J.T. Baker Ethyl acetate J.T. Baker Ethylenediaminetetraacetic acid (EDTA) Honeywell Ferric ammonium citrate Merck Fetal bovine serum (FBS) FBS, JRH Bioscience, Lenexa, KS Formic acid Sigma- Aldrich Fructose Carl Roth Glucose Carl Roth Glycerol Carl Roth HEPES ((4-(2-hydroxyethyl)-1- Carl Roth piperazineethanesulfonic acid) Hydrochloric acid (HCL) Carl Roth Inositol (Ino) Merck

Iron(II) sulfate (FeSO4 x 7 H2O) Honeywell

2 Materials and Methods

Iron(III) chloride (FeCl3 x 6H2O) Merck

Iron(III) citrate (FeC6H6O7 x H2O) Merck Iron(III) ethylenediaminetetraacetic acid (Fe Fluka EDTA) Jump Start Taq Ready Mix Sigma- Aldrich

K2HPO4 Merck

KH2PO4 Carl Roth L-Arginine Panreac Appli Chem L-Asparagine Panreac Appli Chem L-Glutamic acid Merck L-Isoleucine Fluka L-Methionine Panreac Appli Chem L-Tyrosine Merck Malt Extract Carl Roth Meat Extract Carl Roth Molybdatophosphoric acid spray solution for TLC Merck Molybdenum blue spray reagent Sigma

MgSO4 x 7H2O Carl Roth

MnCl2 x 4H2O Merck NaCl Carl Roth Nalidixic acid Carl Roth α-Naphtol Merck α-Naphtol spray reagent composition: 15% α-naphtol in ethanol (10.5 ml)

Concentrated H2SO4 (6.2 ml) Ethanol (40.5 ml) Deionized water (4 ml) Ninhydrin spray reagent Sigma Peptone Becton Dickinson Peptone S Marcor Phytone peptone Carl Roth Potassium chloride (KCl) Sigma- Aldrich Proteose Peptone Becton Dickinson Reef salt “Coral Ocean” ATI, Hamm Sodium glycerophosphate Fluka Sodium thiosulfate-5-hydrate Merck Soluble starch Carl Roth Sucrose Merck

2 Materials and Methods

Sulfuric acid (H2SO4) Merck Water (nuclease free) Carl Roth Yeast extract Carl Roth

ZnSO4 x 7H2O Merck

2.1.3 Source of samples Table 6. List of samples collected from Indonesia for Actinobacteria isolation.

Sample code Location of source Type of sample 1808 Malang, East Java Soil 1899 (FK1) Forest low altitude, Kendari, Southeast Soil Sulawesi 1900 (FK2) Forest low altitude, Kendari, Southeast Soil Sulawesi 1901 (FK3) Forest low altitude, Kendari, Southeast Soil Sulawesi 1902 (FK4) Forest low altitude, Kendari, Southeast Soil Sulawesi 1903 (MK5) Mangrove, Kendari, Southeast Sulawesi Soil 1904 (MK6) Mangrove, Kendari, Southeast Sulawesi Soil 1905 (MK7) Mangrove, Kendari, Southeast Sulawesi Soil 1906 (MK8) Mangrove, Kendari, Southeast Sulawesi Soil 1907 (GC9) Cimahi, West Java Soil 1929 Beach, Bali Sand 1930 Beach, Bali Dead barnacle 1931 Beach, Bali Algae red 1932 Beach, Bali Sand from reef 1933 Beach, Bali Sand 1934 Beach, Bali Sand 1935 Beach, Bali Sand 1936 Beach, Bali Green algae 1937 Beach, Bali Brown algae 1938 Beach, Bali Sand from reef 1939 Beach, Bali Sand from reef 1940 Beach, Bali Grass 1941 Beach, Bali Sand from reef 1942 Beach, Bali Sand

2 Materials and Methods

1943 Beach, Bali Red stone from reef 1944 Beach, Bali Algae 1945 Cultural Park, Bali Sand 1946 Cultural Park, Bali Sand 1947 Cultural Park, Bali Sand 1948 Cultural Park, Bali Sand 1949 Cultural Park, Bali Soil 1950 Cultural Park, Bali Soil 1951 Cultural Park, Bali Soil 1952 Cultural Park, Bali Soil 1953 Cultural Park, Bali Soil 1955 Lava beach, Bali Black sand 1956 Lava beach, Bali Black sand 1957 Lava beach, Bali Black sand 1958 Lava beach, Bali Black sand 1959 Lava beach, Bali Green algae 1960 Lava beach, Bali Black sand 1961 Lava beach, Bali Dead wood 1962 Lava beach, Bali Algae 1963 Beach, Bali Sand 1964 Botanical Garden, Bali Soil with plant residue 1965 Botanical Garden, Bali Soil with plant residue 1966 Botanical Garden, Bali Soil with plant residue 1967 Botanical Garden, Bali Soil with plant residue 1968 Botanical Garden, Bali Soil with plant residue 1969 Botanical Garden, Bali Soil with plant residue 1970 Botanical Garden, Bali Bark 1971 Botanical Garden, Bali Termite soil 1972 Botanical Garden, Bali Lichen grey 1973 Botanical Garden, Bali Fern residues 1974 Botanical Garden, Bali Swpr 1975 Botanical Garden, Bali Soil with moss 1976 Ecology Park, Bogor, West Java Dry grass 1977 Ecology Park, Bogor, West Java Bark 1978 Ecology Park, Bogor, West Java Soil 1979 Ecology Park, Bogor, West Java Dead wood 1980 Ecology Park, Bogor, West Java Dry leaves

2 Materials and Methods

1981 Botanical Garden, Bogor, West Java Exuvia Cicada? 1982 Botanical Garden, Bogor, West Java Soil 1983 Botanical Garden, Bogor, West Java Termite soil 1984 Botanical Garden, Bogor, West Java Soil 1985 Botanical Garden, Bogor, West Java Hypoxylon? 1986 Botanical Garden, Bogor, West Java Bark 1987 Botanical Garden, Bogor, West Java, Hypoxylon? 1988 Botanical Garden, Bogor, West Java Bark dimocarpus 1989 Botanical Garden, Bogor, West Java Lake soil 1990 Botanical Garden, Bogor, West Java Lake soil BL Beach, Lampung Sand BLS Beach, Lampung Seaweed BKB Beach, West Kalimantan Sand BP Beach, Papua Sand BB Beach, Bali Sand MKB Mangrove, West Kalimantan Soil 2111 Mangrove, Jakarta Soil 2112 Mangrove, Jakarta Soil 2113 Mangrove, Jakarta Dead wood 2114 Mangrove, Jakarta Bark 2115 Mangrove, Jakarta Soil 2116 Mangrove, Jakarta Bark 2117 Mangrove, Jakarta Dead wood 2118 Mangrove, Jakarta Soil 2119 Mangrove, Jakarta Soil 2120 Mangrove, Jakarta Dead tree fungi 2121 Mangrove, Jakarta Soil 2122 Mangrove, Jakarta Dead wood 2123 Mangrove, Jakarta Soil 2124 Mangrove, Jakarta Soil 2125 Mangrove, Jakarta Soil 2126 Mangrove, Jakarta Dead leaves 2127 Mangrove, Jakarta Soil 2128 Mangrove, Jakarta Soil 2129 Mangrove, Jakarta Soil 2130 Botanical Garden, Bogor Wood flour 2131 Botanical Garden, Bogor Dead roots

2 Materials and Methods

2132 Botanical Garden, Bogor Moss 2133 Botanical Garden, Bogor Soil 2134 Botanical Garden, Bogor Dead wood 2135 Botanical Garden, Bogor Wood chips 2136 Botanical Garden, Bogor Dead wood 2137 Botanical Garden, Bogor Soil 2138 Botanical Garden, Bogor Bark 2139 Botanical Garden, Bogor Soil 2140 Botanical Garden, Bogor Moss 2141 Botanical Garden, Bogor Soil 2142 Botanical Garden, Bogor Soil 2143 Botanical Garden, Bogor Soil 2144 Botanical Garden, Bogor Soil 2145 Botanical Garden, Bogor Soil 2146 Botanical Garden, Bogor Soil 2147 Botanical Garden, Bogor Soil 2148 Botanical Garden, Bogor Dead roots

2.1.4 Organisms Table 7. Actinobacteria strains from Research Center for Biotechnology LIPI-Indonesia.

Strain Location of source BLH 12-3 Bitung, North Sulawesi, Indonesia DHE 2-1 Enggano Island, Bengkulu, Indonesia DHE 9-4 Enggano Island, Bengkulu, Indonesia MAE 1-3 Enggano Island, Bengkulu, Indonesia MAE 1-11 Enggano Island, Bengkulu, Indonesia SHP 1-2 Enggano Island, Bengkulu, Indonesia SHP 1-4 Enggano Island, Bengkulu, Indonesia SHP 1-5 Enggano Island, Bengkulu, Indonesia SHP 1-6 Enggano Island, Bengkulu, Indonesia SHP 2-2 Enggano Island, Bengkulu, Indonesia SHP 2-4 Enggano Island, Bengkulu, Indonesia SHP 2-5 Enggano Island, Bengkulu, Indonesia SHP 6-2 Enggano Island, Bengkulu, Indonesia SHP 6-3 Enggano Island, Bengkulu, Indonesia SHP 6-4 Enggano Island, Bengkulu, Indonesia

2 Materials and Methods

SHP 6-5 Enggano Island, Bengkulu, Indonesia SHP 6-6 Enggano Island, Bengkulu, Indonesia SHP 7-1 Enggano Island, Bengkulu, Indonesia SHP 7-3 Enggano Island, Bengkulu, Indonesia SHP 7-5 Enggano Island, Bengkulu, Indonesia GKRL-2 Lampung, Indonesia GKRL-3 Lampung, Indonesia GKRL-4 Lampung, Indonesia GBSL-9 Lampung, Indonesia

Table 8. Tested organisms for antimicrobial, antinematode, and cytotoxic activity.

Organism Code or comment Microbes Escherichia coli DSM 1116 Escherichia coli TolC Deficient of TolC (the outer membrane channel for multidrug efflux) Chromobacterium violaceum DSM 30191 Pseudomonas aeruginosa DSM 19882

Staphylococcus aureus Obtained from PD Dr. Markus Bischoff, Newman Saarland University Hospital, Homburg Bacillus subtilis DSM 10 Micrococcus luteus DSM 1790 Mycobacterium smegmatis ATCC 700084

Mucor hiemalis DSM 2656 Pichia anomala DSM 6766 Candida albicans DSM 1665 Mammalian cells A-431 Human epidermoid carcinoma A-549 Human lung carcinoma Huh-7.5 Human hepatocellular carcinoma cell HUVEC Human umbilical vein endothelial cell KB-3-1 Human cervix carcinoma L-929 Murine fibroblast MCF-7 Human breast adenocarcinoma PC-3 Human prostate carcinoma

2 Materials and Methods

SK-OV-3 Human Caucasian ovary adenocarcinoma Nematode Caenorhabditis elegans Non-parasitic roundworm

2.1.5 Kits and enzymes Table 9. The list of kits and enzymes used in the experiment.

Kits or enzymes Usage Manufacturer Invisorb® Spin Plant Mini DNA extraction Stratec/Invitek Kit NucleoSpin® Microbial DNA DNA extraction Macherey-Nagel NucleoSpin® Gel and PCR PCR cleaning Macherey-Nagel Clean-up JumpStart™ Taq PCR Sigma-Aldrich ReadyMix™ Nuclease P1 from Penicillium DNA digestion Sigma-Aldrich citrinum Lysozyme from chicken egg Peptidoglycan lysis Sigma-Aldrich white Proteinase K Protein digestion in DNA Carl Roth extraction Bacterial alkaline phosphatase Removing 3 and 5 Thermo Fisher Scientific phosphates from DNA and RNA API® ZYM Semiquantitation of BioMérieux enzymatic activities API® Coryne 24-hour identification of BioMérieux Corynebacteria and coryne- like organisms 2.1.6 Primers Some primers are used for 16S rRNA analysis and multi locus sequence analysis (MLSA) (Table 10 and Table 11).

Table 10. The list of primers used for 16S rRNA analysis.

Primer Sequence (5’ -> 3’) Length (nt) Positiona Reference F27 AGA GTT TGA TCM TGG 20 8-27 122,148 CTC AG R518 CGT ATT ACC GCG GCT 20 518-537 149 GCT GG F1100 YAA CGA GCG CAA CCC 15 1100-1114 150 R1100 GGG TTG CGC TCG TTG 15 1100-1114 150

2 Materials and Methods

R1492 TAC GGY TAC CTT GTT 22 1492-1513 122,148 ACG ACT T R1525 AAG GAG GTG ATC CAG 20 1522-1541 150 CCG CA a Position number refers to the 16S sequence of E. coli rrnB (GenBank J01695128).

Table 11. The list of primers used for multi locus sequence analysis or MLSA.

Gene Primer Sequence (5’ -> 3’) Length (nt) Reference

atpD atpDPF GTC GGC GAC TTC ACC AAG GGC 36 151 (amplification) AAG GTG TTC AAC ACC atpDPR GTG AAC TGC TTG GCG ACG TGG 38 151 (amplification) GTG TTC TGG GAC AGG AA atpDF ACC AAG GGC AAG GTG TTC AA 20 151 (sequencing) atpDR GCC GGG TAG ATG CCC TTC TC 20 151 (sequencing) gyrB gyrBPFA TC GAG GGT CTG GAC GCG GTC 40 131,152 (amplification) CGC AAG CGA CCC GGT ATG TA gyrBPAR GAA GGT CTT CAC CTC GGT GTT 36 131,152 (amplification) GCC CAG CTT CGT CTT gyrBFA GCA AGC GAC CCG GTA TGT AC 20 131,152 (sequencing) gyrBRA GAG GTT GTC GTC CTT CTC GC 20 131,152 (sequencing) recA recAPF CCG CRC TCG CAC AGA TTG AAC 29 151 (amplification) GSC AAT TC recAPR GCS AGG TCG GGG TTG TCC TTS 32 151 (amplification) AGG AAG TTG CG recAF ACA GAT TGA ACG GCA ATT CG 20 151 (sequencing) recAPR2 GCS AGR TCG GGG TTG TCC TTS 32 152 (sequencing) AGG AAG TTS CG rpoB rpoBPF GAG CGC ATG ACC ACC CAG GAC 29 151 (amplification) GTC GAG GC rpoBPR CCT CGT AGT TGT GAC CCT CCC 30 151 (amplification) ACG GCA TGA rpoBF1 TTC ATG GAC CAG AAC AAC C 19 151 (sequencing) rpoBR1 CGT AGT TGT GAC CCT CCC 18 151 (sequencing)

2 Materials and Methods trpB trpBPF GCG CGA GGA CCT GAA CCA CAC 41 151 (amplification) CGG CTC ACA CAA GAT CAA CA

trpBPR TCG ATG GCC GGG ATG ATG CCC 40 151 (amplification) TCG GTG CGC GAC AGC AGG C trpBF GGC TCA CAC AAG ATC AAC AA 20 151 (sequencing) trpBR TCG ATG GCC GGG ATG ATG CC 20 151 (sequencing)

2.1.7 Equipment Table 12. Equipment used in this work.

Equipment Manufacturer Centrifuge Eppendorf Centrifuge 5804 R Centrifuge Eppendorf Centrifuge 5427 R Clean Bench Thermo Scientific Type MS 2020 1.2

CO2 incubator Thermo Scientific Heracell 150i CO2 Incubator HPLC Agilent 1260 Series; Aligent technology, USA HPLC Aligent 1100 series; Aligent technology, USA HPLC column XBrigde® C-18 3.5 µm, 2.1 mm x 100 mm, Waters Incubator Hereus Instruments Function Line Light microscope Zeiss Axio Sc pie. A1 microscope MS (HRESIMS) MaXis ESI-TOF-MS spectrometer (Bruker) equipped with an Agilent 1260 series RP-HPLC system Multichannel pipette RAININ 8-Kanal-Pipette ED P3 Plus 100-1200 µL N2 dryer (plates) MiniVap (porvair science) Photometer IMPLEN Nano Photometer UV / VIS Spektralphotometer Pipettes Eppendorf Research plus Rotary evaporator Heidolph Laborata 4003 Shaker Pilot-Shake System Kühner RC-6-U Shaker (plates) Heidolph Titramax 1000 Thermocycler Eppendorf Thermocycler Mastercycler gradient

2 Materials and Methods

Thin layer chromatography (TLC) plates Merck cellulose Thin layer chromatography (TLC) plates Merck silica gel 60 UV detector Herolab RH-5.1 darkroom hood + B- 1393-3K7N camera DNA sequencer 96-capillary-system from Applied Biosystems (ABI), 3730xl DNA Analyzer.

2.2 Methods 2.2.1 Sampling The samples were taken by the members of the GINAICO (German-Indonesian Anti- Infective Cooperation) team: Dr. Tjandrawati Mozef, Dr. Kathrin I. Mohr, Dr. Enge Sudarman, and Senlie Octaviana from 2015 until 2017. The locations range from the western part of Indonesia until the eastern part of Indonesia such as Lampung in Sumatra Island; West Kalimantan in Kalimantan Island; Jakarta, Bogor, Cimahi, and Malang in Java Island; Bali Island; Kendari in Sulawesi Island; Papua Island (Figure 5).

2.2.2 Isolation of Actinobacteria One gram of samples was heated at 60°C for 30 minutes to eliminate all the vegetative cells. After the temperature decreased, ten milliliters of sterile water were added to the samples. The mixtures then were serially diluted until a dilution of 1:1000. The samples were plated on agar medium 5336 supplemented with cycloheximide 100 (µg/ml) as antifungal agent153 and incubated for 7-21 days at 30°C.

2.2.3 Extract Production The precultures were cultivated in 100 ml of GYM or GYM+ASW medium in a 250 mL flask for five days at 30°C and 160 rpm on a rotary shaker then were transferred 1:10 to 100 mL of SYP, 5254, 5295, 5254+ASW, and 5294+ASW for metabolite production. The cultures were incubated further for 5-7 days at 30°C and 160 rpm. Afterward, 20 ml of the cultures were extracted with 20 ml of ethyl acetate and were centrifuged at 9000 rpm for 10 min to separate the phases between the two immiscible solvents. The upper phase was then transferred and evaporated with reduced pressure at 40°C. The extract was re-dissolved in 1 ml of ethyl acetate: acetone: methanol (1:1:1) 138,154.

2 Materials and Methods

Figure 5. Map of Indonesia with the sampling sites.The sampling locations are marked with a red circle. Credit picture from https://pasarelapr.com/images/map-of-indonesian- island/map-of-indonesian-island-6.png

2.2.4 Analysis of 16S rRNA sequences Genomic DNA extraction was performed by using Invisorb Spin Plant Mini Kit (250) (Stratec Molecular, Germany). From the well-grown cell suspension, 500 µl were taken and centrifuged for 2 minutes at 11,000 rpm. The supernatant was discarded and the cell pellet was mixed with 100 µL of lysis buffer. The mixture was then incubated at 95 °C for 5 min and added again with 300 µL lysis buffer. Afterward, 20 µL protein kinase K were added and incubated for 30 min at 65°C. The remaining steps were conducted following the manufacturer’s instruction.

Amplification of 16S rRNA genes and the purification of the PCR product were carried out using the methods described by Mohr et al.155. Two primers were used matching most of the known eubacterial orders on the positions 27 (forward) and 1492 (reverse) or 1525 (reverse). The reaction volume (50 µl) was created containing water (22 µl), primers (1 µl; 10 µM each), “Jump Start Ready Mix” or JSRM (25 µl) and template DNA (1 µl). The JSRM is a mixture of JumpStart Taq DNA polymerase, 99% pure deoxynucleotides and buffer in an optimized reaction concentrate. The PCR reaction was conducted in a Mastercycler Gradient (Eppendorf, Hamburg, Germany). The condition of reaction included: initial denaturation at 95°C (5 min); 34 cycles of denaturing at 94°C (30 s); annealing at 52°C (30 s); elongation at 72°C (120 s); final elongation at 72°C (10 min).

2 Materials and Methods

The PCR product was checked on the agarose gel (0.8%) and purified using the NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Germany) following the manufacturer’s protocol. The DNA sequencing was performed by using 96-capillary- system from Applied Biosystems (ABI), 3730xl DNA Analyzer. The primers for sequencing were F27, R518, F1100, R1100, and R1492 or R1525.

Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarities were performed using EzTaxon-e server (http://www. ezbiocloud.net/taxonomy)156 and the sequences of the strains were aligned using the CLUSTAL W algorithm157 from the MEGA X software package158. Phylogenetic analysis was achieved using the maximum-likelihood159, maximum-parsimony160 and neighbour-joining161 algorithms from MEGA X158. The topologies of the inferred trees were evaluated by bootstrap analyses162 based on 1000 replicates.

2.2.5 Morphology and melanin production The morphological characteristics of the strains and the ability to produce melanin were examined by incubating them on ISP2 (International Streptomyces Project 2), ISP3, ISP4, ISP5, ISP6, ISP7 medium, SSM+T (synthetically Suter medium with tyrosine), and SSM-T (synthetically Suter medium without tyrosine)102,163 after 14 days at 30°C. The colors of aerial and substrate mycelium, as well as the diffusible pigments, were determined by comparison with the RAL-code (https://www.ral- farben.de)138. Spore chain morphology and spore-surface ornamentation were observed after 14-30 days at 30°C on ISP 3 medium102. A block of agar (1 cm x 1 cm) containing bacteria with the spores was fixed in glutaraldehyde solution (5%)164. The sample was then critical-point-dried and gold–palladium-sputtered. Afterward, the morphology of the spores was observed by a Zeiss Merlin field emission scanning electron microscope (FESEM) with an Everhart-Thornley SE-detector and an Inlens- SEM detector in a 25:75% ratio applying the SEMSmart software version 5.05109. SEM analysis was done by Prof. Dr. Manfred Rhode, HZI Braunschweig.

2.2.6 Physiological and biochemical characteristic 2.2.6.1 Growth at different temperature and pH

Growth at different temperatures (15, 20, 25, 30, 37 and 44°C) on GYM medium agar and pH range (pH 2, 3, 4, 5, 6, 7, 8, 9 and 10) on ISP 2 medium liquid were observed after incubation for 14 days.

2 Materials and Methods

2.2.6.2 Carbon utilization

Carbon utilization was studied on basal medium for carbohydrate utilization or ISP 9 medium102 containing 10 different carbon sources (1 %, w/v) such as glucose, arabinose, sucrose, xylose, inositol, mannitol, fructose, rhamnose, raffinose, and cellulose. For this study, 12-well flat bottom plate was used.

2.2.6.3 Sodium chloride tolerance

The sodium chloride tolerance study was based on the method of Kutzner et al.165 with different concentration of NaCl: 0%, 2.5%, 5.0%, 7.5%, and 10%. In this examination, the 6-well flat bottom plate was employed.

2.2.6.4 Enzymatic assay

The enzyme profiles were studied by using API ZYM166 and API Coryne strips167. At least five days old liquid culture of the bacteria were used for this study. The enzymes that can be detected with API ZYM and API Coryne system are listed in Table 13.

Table 13. The activities that can be analyzed by API ZYM and API Coryne.

API ZYM API Coryne Alkaline phosphatase Nitrate reduction Butyrate esterase (C4) Pyrazinamidase Caprylate esterase lipase (C8) Pyrrolidony arylamidase Myristate lipase (C14) Alkaline phosphatase Leucine arylamidase β-glucuronidase Valine arylamidase β-galactosidase Cystine arylamidase α-Glucosidase Trypsin N-acetyl-β-glucosamidase α-Chymotrypsin Esculin (β-glucosidase) Acid phosphatase Urease Naphtol-AS-BI-phosphohydrolase Gelatine (hydrolysis) α-Galactosidase Glucose fermantation β-Galactosidase Ribose fermentation β-Glucoronidase Xylose fermentation α-Glucosidase Mannitol fermentation β-Glucosidase Maltose fermentation N-acetyl-β-glucosaminidase Lactose fermentation

2 Materials and Methods

α-Mannosidase Sucrose fermentation α-Fucosidase Glycogen fermentation

2.2.6.5 Antibiotic susceptibility

Antibiotic susceptibility was evaluated by the disc-diffusion plate method137 using antibiotic discs on ISP 2 agar medium incubated at 30°C for 7 days. One loop of bacteria from the agar plate or 100 µl of 5-7 days old liquid culture were diluted with 1 ml sterile water and 100 µl of the suspension was plated on the ISP 2 agar medium. Eight antibiotic discs were used: ampicillin (10 µg/disc), erythromycin (15 µg/disc), gentamycin 30 (µg/disc), tetracycline (30 µg/disc), vancomycin (30 µg/disc), cefotaxime (30 µg/disc), rifampicin (5 µg/disc), and penicillin G (6 µg/disc).

2.2.7 Chemotaxonomy 2.2.7.1 Freeze-dried cells preparation

For chemotaxonomy study, freeze-dried cells were used instead of the wet biomass to ease the extraction of chemical substances from the cells. The biomass of a 5-7 days old grown culture in the medium GYM, ISP2 or TSB was collected by centrifuge at 9000 rpm for 10 minutes. The pellet was washed three times with demineralized water and centrifuged three times at 9000 rpm for 10 minutes. The wet biomass was freeze- dried for two days.

2.2.7.2 Cell wall amino acid analysis

The cell wall amino acid analysis was carried out as described by Staneck and Roberts168. Freeze-dried cells (3 mg) and 200 µl of 20% HCl or 6N HCl were added to a 4 ml brown glass vial. The vial was tightly closed with the lid and heated at 100°C for 18h. The mixture was cooled down and filtered by Whatman filter paper. The filtrate was evaporated at 100°C. Demineralized water (500 µl) was added and the liquid was dried again at 100°C to remove the remains of HCl. The dried extract was dissolved with 100 µl demineralized water. The amount of 2 µl was applied for TLC (Thin Layer Chromatography) analysis using an aluminium-backed cellulose plate. The mobile phase was methanol- demineralized water -6 N HCl-pyridine (80:26:4: 10, vol/vol). Standard amino acid (1 µl, 5 mg/ml) was diaminopimelic acid. Spots of amino acids were visualized by spraying with ninhydrin and heating at 100°C for 3 min.

2 Materials and Methods

2.2.7.3 Whole-cell sugar analysis

The whole-cell sugars analysis were prepared following the method of Staneck and 168 Roberts . Freeze-dried cells (25 mg) and 1.5 ml of 2.5% H2SO4 or 1N H2SO4 were added to a 4 ml brown glass vial. The vial was tightly closed with the lid and heated at 100°C for 2 h. After cooling down, the hydrolysate was transferred to a 15-ml conical centrifuge tube, and saturated barium hydroxide (Ba(OH)2. H2O = 3 gr/10 ml) was added carefully until the pH reached 5.2 and 5.5 (determined with pH paper). The precipitate was removed by centrifugation (9000 rpm, 5 min) and discarded. The supernatant fluid was evaporated at 35°C with nitrogen flow, and the residue was redissolved with 100 µl demineralized water. The amount of 2 µl was applied for TLC analysis using aluminium-backed cellulose plate. The mobile phase was n-butanol- demineralized water-pyridine-toluene (10:6:6:1, vol/vol). Two standards containing mixtures of sugar were employed because of the closely Rf value (retention factor) between the sugar. The first mixed sugar standard (0.5 µl, 10 mg/ml each) contained galactose, arabinose, and xylose. The second mixed sugar standard (0.5 µl, 10 mg/ml each) comprised rhamnose, mannose, glucose, and ribose. Spots of sugars were visualized by spraying with acid aniline phthalate and heating at 100°C for 5 min.

2.2.7.4 Menaquinone analysis

Menaquinones were extracted following the procedure performed by Minnikin et al.169. Freeze-dried cells (100 mg) were transferred into polytetrafluoroethene capped tube. The amount of 4 ml methanol-0.3% aqueous NaCl (90:10) and 4 ml petroleum ether were added to the tube. The suspension was mixed on tube rotator for 30 min. The upper phase was transferred to evaporator tube. The same amount of new petroleum ether was added again to the lower phase and mixed on tube rotator for 30 min. The upper layers were combined and evaporated at 30°C with reduced pressure until the volume remains about 1 ml. The extract was transferred to the 4 ml brown glass vial and dried at 30°C under N2 flow. The dry extract was re-dissolved with 100 µl acetonitrile-isopropanol (65:35). The menaquinones were analyzed by high- performance liquid chromatography103 equipped with diode-array detection and mass spectrometry (HPLC-DAD-MS). Here, high-resolution electron spray ionization mass spectrometry (HR-ESI-MS) data were recorded on a MaXis ESI-TOF-MS spectrometer (Bruker) equipped with an Agilent 1260 series RP-HPLC system. The HPLC system consisted of XBridge C18 column 2.1 x 100 mm, 1.7µm; solvent A was

2 Materials and Methods isopropanol and solvent B was acetonitrile. The gradient system was 100% B for 3 min, 35% B in 5 to 15 min, and 50% B in 16-20 min. The flow rate was 0.6 mL/min, temperature of column was 40°C, and the UV-detection was at 270 nm. Molecular formula was calculated using the Smart Formula algorithm including the isotopic pattern (Bruker).

2.2.7.5 Polar lipid analysis

The polar lipids were extracted by the method of Minnikin et al.170 and identified by two-dimensional thin-layer chromatography as described by Collins and Shah111. Freeze-dried cells (200 mg) were transferred into a polytetrafluoroethene capped tube. The amount of 8 ml Chloroform-MeOH (2:1) was added to the tube and mixed on tube rotator for 16 h. The suspension was filtered with filter paper and washed 2 times with 2 ml Chloroform-MeOH (2:1). The filtrate was combined to evaporation flask (25 ml volume size) and was evaporated with reduced pressure at 30°C. The extract was redissolved with 200 µl Chloroform-MeOH (2:1) and transferred to 0.3 ml brown glass vial.

Examination of polar lipids was conducted by two-dimensional thin-layer chromatography (TLC) using HPTLC Kieselgel 60F254 (Merck) plate (10 x 10 cm). The two‐dimensional thin‐layer chromatography was applied in this analysis. The first dimension was with chloroform/methanol/water (65:25:4 by volume) and in the second dimension was with chloroform/methanol/ acetic acid/water (80:12:15:4 by volume). For detecting all lipids, 10% molybdophosphoric acid in ethanol was sprayed to the plate and heated at 140°C for 15 min. Observation of specific lipid was carried out by spraying different reagents. Ninhydrin solution was used for detecting free amino groups (100°C, 4 min), molybdenum blue for phosphate-containing lipids, α- naphtol-H2SO4, anisaldehyde spray solution reagent (100°C, 10 min) for sugar- containing lipids, and Dragendorffs reagent for quaternary ammonium groups (Table 5).

2.2.7.6 Fatty acid analysis

Fatty acids were extracted, methylated and analyzed using Sherlock Microbial Identification (MIDI) system and the ACTIN version 6 database171. Freeze-dried cells (10 mg) were transferred into 13 x 100 culture tube. The amount of 1 ml saponification reagent (45 g NaOH, 150 ml methanol, 150 ml distilled water) was added to the tube.

2 Materials and Methods

The tube was sealed with teflon lined cap, vortexed and heated in a boiling water bath for 5 minutes. It was vortexed again for 5-10 minutes and returned to the boiling water bath to accomplish the 30-minute heating. The mixture was cooled into room temperature and added by 2 ml methylation reagent (325 ml HCl 6N, 275 ml methanol). The tube was capped, briefly vortexed, and heated for 10 minutes at 80°C. The mixture was cooled down into room temperature and added by 1.25 ml extraction reagent (200 ml hexane and 200 ml methyl tert.-butyl ether). The tube was recapped and tumbled on a rotator for 10 minutes. The tube then was uncapped and the aqueous phase (lower part) was pipetted out and discarded. About 3 ml of washing reagent (10.8 g NaOH dissolved in 900 ml distilled water) was added to the organic phase. The tube was recapped and tumbled for 5 minutes. About 2/3 of the organic phase (upper part) was pipetted into a GC vial for analysis. GC analysis was carried out with the conditions: using 25 m x 0.2 mm phenyl methyl silicone fused silica capillary column, the temperature program increased from 170°C to 270°C at 5°C per minute, employing flame ionization detector (FID), the carrier gas was hydrogen and the “make up” gas was nitrogen. Fatty acid analysis was conducted by Gabriele Pötter and Dr. Richard Hahnke, Leibniz Institut DSMZ-Braunschweig.

2.2.8 G+C (guanine + cytosine) contents determination Genomic DNA for G+C contents analysis was prepared based on procedure conducted by Rong and Huang172 with some modification. Twenty milliliters of well-grown culture were taken, centrifuged, washed three times with demineralized water and suspended in 10 ml STE buffer (75 mM NaCl, 25 mM Tris–HCl, 25 mM EDTA, pH 8.0) containing 5 mg lysozyme. Glass beads were added to the suspension and the mixture was vortexed to homogenize the cells. Afterwards, the suspension was incubated for 1 h at 37oC in the tube rotator. The cells were completely lysed by adding 0.2 ml 20% (w/v) SDS and the lysate was further incubated at 55oC for 2 h in the tube rotator. The DNA was extracted with the addition of chloroform (1:1, by volume) and the tube was centrifuged 9000 rpm for 10 minutes. The polar part (upper layer) was transferred carefully to the new tube and was added by 0.1 volume of 3 M sodium acetate (pH 5.5). The genomic DNA was precipitated with the addition 1 volume of isopropanol, rinsed two times with 70% ethanol, dried and dissolved in 1 ml of nuclease-free water.

2 Materials and Methods

The G+C content analysis was carried out adapting the method of Tamaoka and Komagata173 and Mesbah et al.118. DNA solution containing 0.4-1.0 mg/ml in nuclease-free water was heated at 100°C for 10 minutes and cooled rapidly in an ice bath for 5 minutes. The denatured DNA solution was mixed with 16 µl of 30 mM sodium acetate buffer (pH 5.3), 2 µl of 20 mM ZnSO4, and 2 µl of P1 nuclease (1 mg/ml in sodium acetate buffer; 340 U/ml). The mixture was incubated at 50oC for 1 h. Afterwards, 18 µl Tris Buffer 0.1 M pH 8.1 and 2 µl of Bacterial Alkaline Phosphatase (2.5 U/µl) was added to the mixture and incubated further at 65oC for 1 h. The hydrolysate was centrifuged at 11,000 rpm for 5 min and was transferred to 0.3 ml brown glass HPLC vial.

The HPLC system consisted of XBridge C18 column 2.1 x 100 mm, 1.7µm; solvent A was ammonium acetate buffer 5 mM and solvent B was acetonitrile/ ammonium acetate buffer 5 mM (95:5). The gradient system was 100% A to 95% A for 2 min, 95% A to 80% A for 4 min, 80% A to 60% A for 2 min and isocratic condition of 60% A for 2 min. The flow rate was 0.3 mL/min, the temperature of the column was 40°C, and the UV-detection was at 270 nm.

2.2.9 Multilocus sequence analysis (MLSA) for Streptomyces sp. SHP 1-2. Genomic DNA extraction was performed by using Invisorb Spin Plant Mini Kit (250) (Stratec Molecular, Germany). From the well-grown cell suspension, 500 µl were taken and centrifuged for 2 minutes at 11,000 rpm. The supernatant was discarded and the cell pellet was mixed with 100 µL of lysis buffer. The mixture was then incubated at 95°C for 5 min and 300 µL lysis buffer were added. Afterwards, 20 µL protein kinase K was added and incubated for 30 min at 65°C. The remaining steps were conducted following the manufacturer’s instruction.

Five housekeeping genes for multilocus sequence analysis (MLSA), atpD, gyrB, recA, rpoB, and trpB, were used as reported previously by Guo et al.151. The gene atpD is responsible for expressing ATP synthase F1 beta subunit. The gene gyrB is for producing DNA gyrase B subunit. The gene recA is responsible for recombinase A expression. The genes rpoB and trpB are for preparing RNA polymerase beta subunit, and tryptophan synthase beta subunit, respectively130. The partial sequences of the five housekeeping genes were amplified and sequenced by using the primers based on the method of Guo et al.151, Rong et al.130, and Labeda et al.24 (Table 11). The sequences of the five housekeeping genes were concatenated from head to tail and then aligned

2 Materials and Methods using MUSCLE174 in MEGA X158. The phylogenetic relationships were calculated using the maximum-likelihood159, maximum-parsimony160 and neighbour-joining161 algorithms in MEGA X158. The MLSA distance was determined using Kimura 2- parameter distance175. The topologies of the inferred trees were evaluated by bootstrap analyses162 based on 1000 replicates. The MLSA distance 0.007 is suggested previously equivalent to 70% DNA-DNA relatedness value130. That means the MLSA distance greater than 0.007 is considered as different species.

2.2.10 Bioactivity assay 2.2.10.1 Antimicrobial assay

Antimicrobial activity test was conducted by serial dilution in 96-well plates16. All the wells were filled with 150 µL of the microbial suspension (OD600 0.01 for bacteria and 0.05 for fungi). The first row was filled with another 130 µL of the suspension of the test trains. Then 20 µL of the compound were added to the first row and the sample was serially diluted with two-fold dilution to the next row until the last row. In the last row, 150 µL extra were discarded so that the volume of the suspension in all wells are the same (150 µL). The bacterial test strains were Escherichia coli DSM 1116, Escherichia coli TolC, Chromobacterium violaceum DSM 30191, Pseudomonas aeruginosa DSM 19882, Staphylococcus aureus Newman, Bacillus subtilis DSM 10, Micrococcus luteus DSM 1790, and Mycobacterium smegmatis ATCC 700084. The fungal test strains were Mucor hiemalis DSM 2656, Pichia anomala DSM 6766, and Candida albicans DSM 1665. The plates were incubated for 24-48 hours at 30°C or 37°C. A clear well was considered to show inhibition of microbial growth. Inhibition in A-B rows was considered as low activity, C-E rows were medium activity, and F- H rows were strong activity.

2.2.10.2 Nematicidal actitity

The nematicidal activity against Caenorhabditis elegans was determined by as described by Rupcic et al.176. The amount of 500 nematodes/ml in M9 buffer (NaCl 5 g/l, Na2HPO4.2H2O 7.5 g/l, KH2PO4 3 g/l, MgSO4.7H2O 0.25 g/l, pH 7.2) and 66 µl of the tested compound were added in 24-well plate. The plates were then incubated at 20°C for 18 h. The dead and alive worms were counted using a microscope and a mechanic counter.

2 Materials and Methods

2.2.10.3 Cytotoxic assay

In vitro cytotoxicity assay was carried out based on the method of Landwehr et al.154. The cell lines for the assay were L-929 (murine fibroblast), KB-3-1 (human cervix carcinoma), A-549 (human lung carcinoma), PC-3 (human prostate carcinoma), MCF- 7 (human breast adenocarcinoma), A-431 (human epidermoid carcinoma), SK-OV-3 (human Caucasian ovary adenocarcinoma), and HUVEC (human umbilical vein endothelial cell). The cells were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; high glucose) supplemented with 10% FCS (fetal calf serum). Then, 60 µl of serial dilutions of the test compounds were added to 120 µL of suspended cells (50,000 cells/mL) in the wells of 96-well plates. After five days of incubation, the inhibition of propagation (IC50) was determined using an MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The cytotoxic assay was conducted by Wera Collisi, HZI-Braunschweig.

2.2.10.4 Antiviral assay

The antiviral assay was conducted according to Mulwa et al.177. Huh-7.5 cells (human hepatocellular carcinoma cells) stably expressing Firefly luciferase (Huh-7.5 Fluc) were inoculated with RLuc Jc1 reporter viruses and the extracts were given also to the cells. The cells were incubated at 37°C with 5% CO2 supply. After 4 hours of incubation, the inoculum was discarded and the cells were washed with PBS (phosphate buffer saline) and were added with new medium without extracts. The medium for the cells was Dulbecco’s modified minimum essential medium (DMEM, Life Technologies Manchester UK) containing 2 mM glutamine, 1 × minimum essential medium nonessential amino acids (MEM NEAA, Life Technologies), 100 μg/mL streptomycin, 100 IU/mL penicillin (Life Technologies), 5 μg/mL blasticidin and 10% FBS (fetal bovine serum). After 3 days of incubation, the infected cells were lysed. The reporter virus infection and the cell viability was determined by renilla luciferase and firefly luciferase activity, respectively. The measurement of luciferase activity was performed by using Berthold Technologies Centro XS3 Microplate Luminometer. The antiviral assay was conducted by Dimas F. Praditya and Prof. Dr. Eike Steinmann, TWINCORE-Hannover.

2 Materials and Methods

2.2.11 Fermentation, extraction and isolation of secondary metabolites from Streptomyces sp. SHP 1-2 The pre-culture of strain SHP 1-2 was grown in 250 mL Erlenmeyer flasks containing 100 mL GYM medium154 for 5 days at 30oC on a rotary shaker (160 rpm). The inoculum was transferred (1:10, by volume) into 500 mL Erlenmeyer flasks containing 200 mL 5294 medium154. A total of 10 L of liquid medium was cultivated at 30oC for 5 days on the shaker with 160 rpm.

The fermentation broth (10 L) was harvested after 5 days of incubation and was centrifuged at 8500 rpm for 30 minutes. Afterwards, 5% (v/v) XAD-2 polymeric resin suspension was added to the filtrate and was stirred slowly for 16 hours in dark condition. The mycelium was extracted with 500 ml of acetone for three times. The extracts were combined and concentrated at 40°C with reduced pressure until the aqueous residue. This residue then partitioned with n-heptane and ethyl acetate respectively to yield n-heptane extract (63 mg) and ethyl acetate extract (781 mg). The solid residue from the mycelium was extracted again with ethyl acetate, evaporated at 40°C with reduced pressure, re-dissolved with methanol, and partitioned with n- heptane. The methanol extract then evaporated at 40°C under vacuum to give methanol extract (63 mg). The n-heptane extract (63 mg), ethyl acetate extract (781 mg), and methanol extract (63 mg) were combined and fractionated by a silica gel chromatography column eluting with gradient of CH2Cl2-ethyl acetate (from 100:0 to 0:100, by volume) to give eight fractions. Fraction 4 and fraction 7 were introduced separately to preparative reversed-phase HPLC using a C18 column with the gradient system (from 10% Acetonitrile: 90% Water to 100% Acetonitrile, by volume) to yield fraction 4.1 (35 mg) and fraction 7.1 (70 mg). The fermentation, extraction, and isolation of compound were carried out together by Dr. Rizna T. Dewi (RChem LIPI, Serpong), Gian Primahana, and Dr. Enge Sudarman (HZI-Braunschweig).

2.2.12 Fermentation, extraction and isolation of compound from Amycolatopsis sp. 196526CR The pre-culture of strain 196526CR was grown in 250 mL Erlenmeyer flasks containing 100 mL GYM medium154 for 5 days at 30oC on a rotary shaker (160 rpm) and the inoculum was transferred (1:10, by volume) into 250 ml Erlenmeyer flasks containing 100 ml, 500 ml Erlenmeyer flasks containing 200 ml, or 1000 ml Erlenmeyer flasks containing 400 mL 5294HG-S medium (Table 4). A total of 15 L of liquid medium was cultivated at 30oC for 7 days in the shaker 160 rpm.

2 Materials and Methods

The fermentation broth (15 L) was harvested after 7 days of incubation and centrifuged at 8500 rpm for 30 minutes. Afterwards, 5% (v/v) XAD-2 polymeric resin suspension were added to the filtrate and was stirred slowly for 16 hours in dark condition. The mycelium was extracted with acetone: methanol (1:1) for five times. The extracts were combined and concentrated at 40°C with reduced pressure until the aqueous residue. The aqueous residue was partitioned with ethyl acetate to yield ethyl acetate extract (6.1 g). The ethyl acetate extract was re-dissolved with 95% methanol and partitioned with n-heptane and dichloromethane to give n-heptane extract (3.3 g) and dichloromethane extract (551.5 mg). The dichloromethane extract was fractionated by an RP-18 chromatography column eluting with gradient of water (with formic acid 0.1%) and acetonitrile (with formic acid 0.1%) (from 40:60 to 0:100, by volume) to give fraction GRP17-19 (8.7 mg). Fraction GRP17-19 was further purified with RP- 18 chromatography column eluting with gradient of water (with formic acid 0.1%) and acetonitrile (with formic acid 0.1%) (from 50:50 to 0:100, by volume) to yield fraction BDP61-63 (1.5 mg).

3 Results

3 Results 3.1 Isolated Actinobacteria, 16S rRNA gene characterization, and bioactivity of the extracts There were 196 Actinobacteria strains isolated from Indonesian samples. Twenty four of them were already isolated in Indonesia by Dr. Shanti Ratnakomala (Research Center of Biotechnology-LIPI). From 196 isolates, 84 strain were analyzed for their 16S rRNA gene during this work. There were 59 isolates characterized as Streptomyces and 25 isolates were non-Streptomyces. Some selected strains with with low percentage similarity from the closest type strains are shown in Table 14. There were 82 strains used for extract production and tested for antimicrobial activity. Some of the extracts, totally 58 extracts, had interesting antimicrobial activity with moderate and strong level. Some selected strains that can produce extracts with moderate and strong antimicrobial activity can be seen in Table 15. From 101 extracts that were tested for antiviral activity, only 46 extracts had promising antiviral activity with moderate (20-50% infectivity), strong (3-20% infectivity) and very strong level (< 3% infectivity). The nontoxic extracts that have strong antiviral activity against HCV are listed in Table 16.

Table 14. Some isolated Actinobacteria with low percentage similarity from the closest species based on 16S rRNA gene

No. Strain Closest type strain Similarity Completeness (%) of the sequence (%) 1 9BLSSO Streptomyces lanatus 97.03 99.9 2 195336CR Mycobacterium palauense 98.47 100 3 194938CR Kibdelosporangium 98.74 100 banguiense 4 195105 Streptomyces cyaneus 98.77 50.7 5 195107 Streptomyces cyaneus 98.77 50.6 6 195227GnCR Streptomyces filipinensis 98.81 58.0 7 DHE 9-4 Streptomyces spongiae 98.83 100 8 196526CR Amycolatopsis thermalba 98.85 100 9 190122BCR Streptomyces roietensis 98.89 58.2 10 190233 Streptomyces glomeratus 98.96 33.3 11 SHP 1-2 Streptomyces 99.03 99.9 viridochromogenes

3 Results

Table 15. Selected strains that can produce extracts with moderate and strong activity against some microbes

No. Strain Production Antimicrobial activity medium 1 SHP 1-2 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) Staphylococcus aureus (moderate) Micrococcus luteus (strong) 2 195105 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) Staphylococcus aureus (moderate) Micrococcus luteus (moderate) Mycobacterium smegmatis (moderate) Fungi Mucor hiemalis (moderate) 3 195107 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) Staphylococcus aureus (strong) Micrococcus luteus (moderate) Mycobacterium smegmatis (moderate) Fungi Mucor hiemalis (moderate) 4 196526CR 5294 Gram-positive bacteria (Amycolatopsis sp.) Bacillus subtilis (moderate) Staphylococcus aureus (moderate)

Fungi Mucor hiemalis (moderate) Candida albicans (moderate) 5 194938CR 5254 Gram-negative bacteria (Kibdelosporangium Escherichia coli TolC (moderate) sp.) Gram-positive bacteria Staphylococcus aureus (moderate) 6 9BLSSO 5294 Gram-positive bacteria (Streptomyces sp.) Staphylococcus aureus (moderate) Fungi Mucor hiemalis (moderate) 7 195227GnCR 5254 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) 8 190233 5294 Fungi (Streptomyces sp.) Mucor hiemalis (moderate)

3 Results

Table 16. List of non-toxic extracts having antiviral activity with strong level against HCV

No. Strain Extract 1 DHE 2-1 DHE 2-1_5254 2 DHE 2-1 DHE 2-1_5294 3 MAE 1-11 MAE 1-11_5294 4 MAE 1-11 MAE 1-11_SYP 5 SHP 1-4 SHP 1-4_5294 6 SHP 2-2 SHP 2-2_5294 7 SHP 6-6 SHP 6-6_5294 8 190231 190231_5294 9 C190221 C190221_5254 10 C194911 C194911_5294 11 C195321A C195321A_5254 12 C195321A C195321A_5294 13 C196921 C19692 _5254 3.2 Polyphasic taxonomy of Streptomyces sp. SHP 1-2 Morphology and melanin production Streptomyces sp. SHP 1-2 grew well on GYM, ISP 2, ISP 3, ISP5, ISP6, ISP7, SSM+T and SSM-T medium agar and moderately on ISP 4 medium agar (Table 17). The morphology of the strain in GYM and ISP 2 medium can be seen in Figure 6. Scanning electron microscopy, after 14 days of growth on ISP 3, showed spiral chains of smooth surface spores (Figure 7).

Table 17. Growth and characteristics of Streptomyces sp. SHP 1-2 cultivated on various agar media after incubation for 14 days at 30 °C

Agar medium Growth Substrate Aerial Soluble mycelium mycelium pigment color color Yeast extract-malt Good Olive brown, Traffic grey B, None extract (ISP 2) ochre yellow signal white Oatmeal (ISP 3) Good Ivory Telegrey 2, None signal white Inorganic salt- Moderate Ivory Telegrey 2, None starch (ISP 4) signal white Glycerol- Good Ivory, light Telegrey 2, None asparagine (ISP 5) ivory signal white Peptone-yeast Good Maize yellow Signal white None extract-iron (ISP 6)

3 Results

Tyrosine (ISP 7) Good Ivory light, Traffic grey A, None ivory signal white Synthetically Suter Good Ocker brown, Signal white None medium with ivory tyrosine (SSM+T) Synthetically Suter Good Ocher brown, Signal white None medium without ivory tyrosine (SSM-T)

Figure 6. Morphology of Streptomyces sp. SHP 1-2 on GYM (left) and ISP 2 medium (right).

Figure 7. Scanning electron micrographs of aerial mycelium and spores of Streptomyces sp. SHP 1-2 after incubation on ISP 3 agar for 14 days at 30 °C.

3.1.1 Chemotaxonomy The cell wall of Streptomyces sp. SHP 1-2 comprised of LL-diaminopimelic acid. The whole-cell sugar analysis of Streptomyces sp. SHP 1-2 suggested that the strain

3 Results contained glucose and xylose. The TLC chromatogram for amino acid of the cell wall and whole-cell sugar analysis can be seen in (Figure 8). The predominant fatty acids of Streptomyces sp. SHP 1-2 were anteiso-C15:0 (29.99%), iso-C16:0 (14.61%), anteiso-C17:0 (11.55%), iso-C15:0 (11.16%) and C16:0 (9.24%) (Figure 9). Its major menaquinones comprised as MK-9(H4) 9.31%, MK-9(H6) 68.03%, MK-9(H8) 22.65% in a ratio of 1:7:2 (Figure 10). The polar lipids were detected as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and five unidentified polar lipids (Figure 11).

Figure 8. TLC chromatogram of whole-cell sugar analysis (left) and analysis amino acid of the cell wall (right) of Streptomyces sp. SHP 1-2. TLC chromatogram of whole-cell sugar analysis (left) and analysis amino acid of the cell wall (right) of Streptomyces sp. SHP 1-2. S1: Standard 1; S2: Standard 2; X: Xylose; A: Arabinose; Gal: Galactose; DPM: Diaminopimelic acid standard; LL: LL- Diaminopimelic acid; Meso: meso- Diaminopimelic acid; SHP 1-2: Streptomyces sp. SHP 1-2.

3.2.1 16S rRNA gene analysis The almost complete 16S rRNA gene sequence of Streptomyces sp. SHP 1-2 (1,509 nucleotides) was determined and deposited under the GenBank accession number MK287949. Based on the result from EzTaxon server (http://www. ezbiocloud.net/taxonomy)156, the strain was known to be closely related to the type strains of Streptomyces viridochromogenes NBRC 3113T (99.03%), Streptomyces malachitofuscus NBRC 13059T (99.03%), and Streptomyces misionensis DSM 40306T (98.96%). Streptomyces sp. SHP 1-2 was also found to form a distinct phyletic line

3 Results from other 18 close species in the phylogenetic tree based on the 16S rRNA gene sequence (Figure 12).

RT Response Ar/H RFact ECL Peak Name Percent 1.629 3.712E+8 0.026t ---- 7.011 SOLVENT PEAK ---- 2.129 255 0.024 ---- 8.003 ---- 2.537 297 0.020 ---- 8.815 ---- 2.890 306 0.023 1.194 9.515 unknown 9.521 0.50 6.738 2374 0.036 0.986 13.618 14:0 ISO 3.17 6.810 364 0.033 ---- 13.671 ---- 7.258 389 0.033 0.976 13.998 14:0 0.51 8.218 8561 0.039 0.962 14.622 15:0 ISO 11.16 8.358 23054 0.039 0.960 14.713 15:0 ANTEISO 29.99 9.565 832 0.041 0.948 15.459 16:1 ISO H 1.07 9.842 11404 0.042 0.946 15.625 16:0 ISO 14.61 10.160 641 0.041 0.943 15.816 16:1 CIS 9 0.82 10.463 7248 0.040 0.941 15.998 16:0 9.24 11.188 1392 0.040 0.937 16.417 16:0 9? METHYL 1.77 11.370 3079 0.042 0.937 16.522 17:1 ANTEISO C 3.91 11.555 2388 0.042 0.936 16.629 17:0 ISO 3.03 11.715 9119 0.042 0.935 16.722 17:0 ANTEISO 11.55 12.005 3258 0.046 0.934 16.889 17:0 CYCLO 4.12 13.071 937 0.040 0.932 17.497 UNKNOWN 17.493 SM 1.18 13.246 2677 0.046 0.932 17.596 UNKNOWN 17.595 SM 3.38

Figure 9. GC chromatogram of fatty acid analysis of Streptomyces sp. SHP 1-2.

3.2.2 Multilocus sequence analysis (MLSA) Based on MLSA analysis resulting from the concatenation of 5 house-keeping genes head to tail, it was determined that Streptomyces sp. SHP 1-2 and Streptomyces fumigatiscleroticus NRRL B-3856 T were very closely related with high significant bootstrap values or by the stability of the relationship when different phylogenetic algorithms are used, such as maximum-likelihood and neighbour-joining analyses (Figure 13). The MLSA distance between Streptomyces sp. SHP 1-2 and S.

3 Results fumigatiscleroticus NRRL B-3856T was 0.058 (Table 18). Streptomyces sp. SHP 1-2 showed to have MLSA distance more than 0.007 with all of the other related species. The similarities of 16S rRNA gene sequence between Streptomyces sp. SHP 1-2 and S. fumigatiscleroticus NRRL B-3856 T was 97.72%.

B m/z + [M+H] 791 m/z + [M+H] m/z [M+H] 789 + 793

Figure 10. Menaquinones detected in Streptomyces sp. SHP 1-2. A: structure of menaquinone-9 (MK-9); B: Chromatogram from LC-MS of detected menaquinones; C: UV Spectrum of menaquinone.

Figure 11. Polar lipid observed in Streptomyces sp. SHP 1-2. DPG: diphosphatidylglycerol; PE: phosphatidethanolamine; PI: phosphatidylinositol; PIM: phosphatidylinositol mannoside; GL: unknown glycolipid; PL: unknown phospholipid; APL: unknown aminophospholipid; AL1-2: unknown aminolipids.

3 Results

Figure 12. Neighbour-joining tree based on 16S rRNA gene sequences (1435 positions in the final dataset) showing relationships between strain SHP 1-2T and the type strains of closely related Streptomyces species. The evolutionary distances were determined using the Tamura-Nei method178. Asterisks indicate branches of the tree that were also found using the maximum-likelihood and maximum-parsimony tree-making algorithms. ML specify nodes that were also recovered using the maximum-likelihood. Numbers at the nodes are percentage bootstrap values based on a neighbour-joining analysis of 1,000 replicates, only values above 50 % are shown. Bar 0.0050 substitutions per nucleotide position.

3.2.3 G+C content and full genome sequencing The G+C content of Streptomyces sp. SHP 1-2 was found to be 73.37% based on HPLC analysis (Figure 14). Full genome analysis based on next-generation sequencing technique (NGS) exhibited that the genome size of the strain was 7,562765 bp.

3.2.4 Physiological and biochemical characteristic Streptomyces sp. SHP 1-2 could grow at 15-37oC (optimum 25-30oC) and pH 6-9 (optimum pH 7). The strain was also able to grow in the presence of 7.5% NaCl, albeit the aerial mycelium was found only until 5% NaCl. Strain SHP 1-2 was sensitive to erythromycin (15 µg/disc), gentamycin 30 (µg/disc), tetracycline (30 µg/disc), vancomycin (30 µg/disc), and rifampicin (5 µg/disc). However it is resistant to ampicillin (10 µg/disc), cefotaxime (30 µg/disc), and penicillin G (6 µg/disc).

3 Results

Streptomyces sp. SHP 1-2 was only able to use glucose as their source of carbon. The results from API ZYM suggested that the strain possessed strong enzymatic activities for phosphatase alkaline, leucine arylamidase, alpha-glucosidase, and N-acetyl-beta- glucoseamidase; however, it had weak activity for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cysteine arylamidase, phosphatase acid, naphtol-AS- BI-phosphohydrolase, beta-galactosidase, and beta-glucosidase. From API Coryne tests, it was found that the strain had positive results for alkaline phosphatase, alpha- glucosidase, N-acetyl-beta-glucoseamidase, esculin (beta-glucosidase), and gelatinase (Table 19).

Figure 13. Neighbour-joining tree based on concatenated partial sequences of the house- keeping genes atpD, gyrB, recA, rpoB and trpB. The evolutionary distances were determined using the Tamura-Nei method178. There were totally 2528 positions in the final dataset. Asterisks indicate branches of the tree that were also found using the maximum-likelihood and maximum-parsimony tree-making algorithms. ML and MP specify nodes that were also recovered using the maximum-likelihood and maximum- parsimony, respectively. Numbers at the nodes are percentage bootstrap values based on a neighbour-joining analysis of 1,000 replicates, only values above 50 % are shown. Bar 0.010 substitutions per nucleotide position.

3 Results

Table 18. MLSA distance between Streptomyces sp. SHP 1-2 and other related strains

Compared strain MLSA distance Streptomyces misionensis NRRL B-3230 0.072 Streptomyces paradoxus NRRL B-3457 0.094 Streptomyces viridochromogenes NRRL B-1511 0.081 Streptomyces lavenduligriseus NRRL ISP-5487 0.073 Streptomyces malachitofuscus NRRL B-12273 0.083 Streptomyces eurythermus NRRL ISP-5014 0.075 Streptomyces phaeoluteichromatogenes NRRL B-5799 0.073 Streptomyces nogalater NRRL ISP-5546 0.078 Streptomyces calvus NRRL B-2399 0.079 Streptomyces flaveolus NRRL B-1334 0.085 Streptomyces leeuwenhoekii NRRL B-24963 0.072 Streptomyces fumigatiscleroticus NRRL B-3856 0.058 Streptomyces griseomycini NRRL B-5421 0.069

dC dG

dA dT

Figure 14. G+C content from the genome of Streptomyces sp. SHP 1-2 based on HPLC anlaysis.

3 Results

Table 19. API ZYM and API Coryne test result for Streptomyces sp. SHP 1-2

API ZYM Activity API Coryne Activity Alkaline phosphatase ++ Nitrate reduction - Butyrate esterase (C4) (+) Pyrazinamidase - Caprylate esterase lipase (C8) (+) Pyrrolidony arylamidase - Myristate lipase (C14) (+) Alkaline phosphatase + Leucine arylamidase ++ β-glucuronidase - Valine arylamidase (+) β-galactosidase - Cystine arylamidase (+) α-Glucosidase + Trypsin - N-acetyl-β-glucosamidase + α-Chymotrypsin - Esculin (β-glucosidase) + Acid phosphatase (+) Urease - Naphtol-AS-BI- (+) Gelatine (hydrolysis) + phosphohydrolase α-Galactosidase - Glucose fermantation - β-Galactosidase (+) Ribose fermentation - β-Glucoronidase - Xylose fermentation - α-Glucosidase ++ Mannitol fermentation - β-Glucosidase (+) Maltose fermentation - N-acetyl-β-glucosaminidase ++ Lactose fermentation - α-Mannosidase - Sucrose fermentation - α-Fucosidase - Glycogen fermentation - ++ more positive result; + positive result; (+) weakly positive result; - negative result.

3.2.5 Phenotypic characteristic between Streptomyces sp. SHP 1-2 and its closest related strains To make more support for separation between species, some phenotypic properties of Streptomyces sp. SHP 1-2 was compared to the very close strains based on its 16S rDNA and MLSA result. Streptomyces malachitofuscus DSM 40332T, Streptomyces viridochromogenes DSM 40110 T and Streptomyces misionensis DSM 40306 T were used as they are very close based on their 16S rDNA. Streptomyces fumigatiscleroticus DSM 43154 T was employed because it is very close according to MLSA result (Table 20).

3 Results

Table 20. Some phenotypic comparison between Streptomyces sp. SHP 1-2 and its closest related strains

Characteristic 1 2 3 4 5 ISP 2 - aerial mycelium Traffic grey B, signal Sparse Grey Blue grey None white ISP 6 - soluble pigment None None Brown Brown None ISP 7 - soluble pigment None Red Black None None Use of carbohydrate Glucose + - + + + Arabinose - + + + (+) Sucrose - - + - - Xylose - - - + ++ Inositol - - + + (+) Mannitol - + (+) + (+) Fructose - + + + (+) Rhamnose - + + - - Raffinose - + - - - API ZYM Phosphatase alcaline ++ ++ ++ ++ ++ Trypsin - (+) + + - Chymotrypsin - (+) + (+) (+) α-Glucosidase ++ + ++ - ++ N-acetyl-beta- ++ ++ ++ - + glucoseamidase α-Mannosidase - ++ ++ ++ ++ API Coryne Nitrate reduction - + - - - Pyrrolidonyl arylamidase - - - - + β-Galactosidase - + - - + Esculin (beta glucosidase) + + - - + Gelatine(hydrolysis) + + + - + ++ more positive result; + positive result; - negative result; (+) weakly positive result; 1: Strain SHP 1-2; 2: S. fumigatiscleroticus DSM 43154 T; 3: S. malachitofuscus DSM 40332; 4: S. viridochromogenes DSM 40110; 5: S. misionensis DSM 40306.

3 Results

3.3 Compounds isolated from Streptomyces sp. SHP 1-2 and their biological activity This experiment was conducted together with Gian Primahana (Ph.D student in the working group “Microbial drugs” of the HZI, Braunschweig, Germany), Dr. Rizna T. Dewi (scientist from Research Center of Chemistry, LIPI, Indonesia), and Dr. Enge Sudarman (scientist in the working group “Microbial drugs” of the HZI, Braunschweig, Germany). There were six compounds isolated from Streptomyces sp. SHP 1-2. Two of them were new compounds (indolactam variant 1 and indolactam variant 2). Four known compounds were Lyngbyatoxin A, Cyclo(phenylalanylprolyl), Indolactam V, and Piericidin A1 (Figure 15).

Cyclo(phenylalanylprolyl)

Lyngbyatoxin A New isolated Compounds (isomers): Indolactam V Indolactam Variant 1 Indolactam Variant 2

Molecular formula:

C17H23N3O3 Molecular weight: 317.39

Piericidin A1

Figure 15. Compounds isolated from Streptomyces sp. SHP 1-2.

Piericidin A1 showed potent antimicrobial activity against Escherichia coli TolC, Chromobacterium violaceum, Bacillus subtilis, and Micrococcus luteus with the MIC values less than 5 µg/ml. Piericidin A1 also had activity in the inhibition of almost all tested mammalian cells. Besides, piericidin A1 was the only isolated compound that possessed antinematode activity. Indolactam variant 1 and indolactam variant 2 showed no activity against all of the tested bacteria, fungi, and nematode. Indolactam variant 1 inhibited moderately the growth of KB-3-1 and PC-3 cells with IC50 8 and 31 µg/ml, respectively. Indolactam variant 2 could inhibit reasonably KB-3-1, PC-3, and SK-OV-3 cell lines with the IC50 3.5, 18, and 25 µg/ml respectively. Lyngbyatoxin A showed antimicrobial activity against some Gram-positive bacteria Staphylococcus aureus, B. subtilis, and M. luteus with the MIC less than 5 µg/ml. Lyngbyatoxin A

3 Results exhibited cytotoxic activity against all of eight mammalian cells; however, no activity was observed against nematode Caenorhabditis elegans (Table 21).

Table 21. Biological activity of compounds isolated from strain SHP 1-2.

Compound Tested orgainsm 1 2 3 4 5 6 Microbes MIC (µg/ml) Escherichia coli > 66.7 ND > 66.7 > 66.7 ND > 66.7 Escherichia coli 2.32 ND > 66.7 > 66.7 ND > 66.7 TolC Chromobacterium 1.16 ND > 66.7 > 66.7 ND > 66.7 violaceum Pseudomonas > 66.7 ND > 66.7 > 66.7 ND > 66.7 aeruginosa Staphylococcus > 66.7 ND > 66.7 > 66.7 ND 4.75 aureus Bacillus subtilis 2.5 ND > 66.7 > 66.7 ND 2.38 Micrococcus luteus 4.65 ND > 66.7 > 66.7 ND 4.75 Mycobacterium > 66.7 ND > 66.7 > 66.7 ND > 66.7 smegmatis Mucor hiemalis > 66.7 ND > 66.7 > 66.7 ND > 66.7 Pichia anomala > 66.7 ND > 66.7 > 66.7 ND > 66.7 Candida albicans > 66.7 ND > 66.7 > 66.7 ND > 66.7 Mamalian cells IC50 (µg/ml) L-929 5.1 ND > 100 > 100 ND 4.1 KB-3-1 0.57 x ND 8 3.5 ND 2 10-3 A-549 ND ND > 100 > 100 ND 0.45 x 10-3 PC-3 0.33 x ND 31 18 ND 1.7 10-3 MCF-7 8.55 x ND > 100 > 100 ND 3.4 10-3 A-431 17 x 10-3 ND > 100 > 100 ND 2.3 SK-OV-3 0.64 x ND > 100 25 ND 5.5 10-3 HUVEC 25 x 10-3 ND ND ND ND 1.9 Nematode LC50 (µg/ml) Caenorhabditis 10.8 > 100 > 100 > 100 > 100 > 100 elegans 1: Piericidin A1; 2: Indolactam V; 3: Indolactam variant 1; 4: Indolactam variant 2; 5: Cyclo (phenylalanyalprolyl); 6: Lyngbyatoxin A; ND: Not Determined.

3 Results

3.4 Taxonomy study and extract analysis of strain MAE 1-11 3.4.1 Morphology and melanin production Growth of isolate MAE 1-11 was well observed on ISP 2, ISP 3, ISP 4, ISP 5, ISP 6, ISP 7, SSM+T and SSM-T medium. The strain can also produce melanin in ISP 6 and SSM+T medium (Table 22). The morphology of the isolate MAE 1-11 in GYM and ISP 2 medium can be seen in Figure 16.

Table 22. Growth and characteristics of strain MAE 1-11 grown on various agar media after incubation for 14 days at 30°C

Agar medium Substrate Aerial mycelium Soluble pigment mycelium color color Yeast extract-malt Maize yellow; Cream None extract (ISP 2) Brown beige Oatmeal (ISP 3) Maize yellow; Grey white; None Saffron yellow Signal white Inorganic salt- starch Sand yellow; Signal white None (ISP 4) Broom yellow Glycerol-asparagine Sand yellow; Signal white None (ISP 5) Lemon yellow Peptone-yeast extract- Yellow olive; Signal white Chocolate brown; iron (ISP 6) olive drab; sepia mahogany brown brown Tyrosine (ISP 7) Brown beige; Silk grey; grey None maize yellow white; signal white Synthetically Suter Olive brown; clay None Brown grey medium with tyrosine brown (SSM+T) Synthetically Suter Maize yellow; None None medium without lemon yellow tyrosine (SSM-T)

3.4.2 16S rRNA gene analysis The almost complete 16S rRNA gene (1,498 nt) was analyzed by EzTaxon server (http://www. ezbiocloud.net/taxonomy)156 and the result suggested that isolate MAE 1-11 was found to be closely related to the type strains of Kitasatospora albolonga NBRC 13465(T) (99.93%), Streptomyces cavourensis NBRC 13026T (99.86%), and Streptomyces bacillaris NBRC 13487T (99.38%). In the phylogenetic tree based on the 16S rRNA gene sequences of strain MAE 1-11 and its closest type strains, the isolate MAE 1-11 formed a distinct subclade and clustered with S. cavourensis NBRC

3 Results

13026T, close to K. albolonga NBRC 13465T and Streptomyces araujoniae ASBV-1T by using Neighbour-Joining method161. However, the subclade consisting of strain MAE 1-11 and S. cavourensis NBRC 13026T were not founded in the tree constructed by using the maximum-likelihood and maximum-parsimony algorithms (Figure 17).

Figure 16. Morphology of strain MAE 1-11 on GYM (left) and ISP 2 medium (right).

Figure 17. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences between strain MAE 1-11 and its closely related neighbours. The evolutionary history was inferred using the Neighbor-Joining method161. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches162. The evolutionary distances were computed using the Kimura 2-parameter method175. There were a total of 1426 positions in the final dataset. Evolutionary analyses were conducted in MEGA X158. Asterisks and filled circles indicate branches of the tree that were also found using the maximum-likelihood and maximum-parsimony tree-making algorithms, respectively.

3 Results

3.4.3 Physiological and biochemical characteristic Strain MAE 1-11 was able to grow in the presence of 10% NaCl; however, the aerial mycelium was found only until 5% NaCl. The strain could be cultivated by using glucose, xylose, mannitol, and fructose (Table 23).

Table 23. Some physiology properties of strain MAE 1-11

Characteristic Observation Observation Use of carbohydrate Use of carbohydrate Glucose + Mannitol + Arabinose - Fructose + Sucrose (+) Rhamnose (+) Xylose + Raffinose (+) Inositol (+) Cellulose - 10%; aerial NaCl tolerance mycelium until 5% API ZYM API ZYM ++ Naphtol-AS-BI- + Phosphatase alkaline phosphohydrolase Esterase (C4) + α-Galactosidase - Esterase Lipase (C8) + β-Galactosidase (+) Lipase (C14) ++ β-Glucuronidase - Leucin arylamidase ++ α-Glucosidase ++ Valine arylamidase ++ β-Glucosidase ++ + N-acetyl-beta- - Cystine arylamidase glucoseamidase Trypsin ++ α-Mannosidase - Chymotrypsin ++ α-Fucosidase - Phosphatase acid + API Coryne API Coryne Nitrate reduction + Gelatine (hydrolysis) + Pyraziamidase - Glucose fermentation - Pyrrolidonyl arylamidase - Ribose fermentation - Alkaline phosphatase + Xylose fermentation - β-Glucuronidase - Mannitol fermentation - β-Galactosidase + Maltose fermentation - α-Glucosidase + Lactose fermentation -

3 Results

N-acetyl -beta - - Sucrose fermentation glucoseamidase Esculin (beta glucosidase) + Glycogen fermentation - Urease + ++ more positive result; + positive result; (+) weakly positive result; - negative result.

3.4.4 Raw extract analysis The raw extract derived from SYP medium was analyzed by LC-HRMS (MAXIS) and the chromatogram result can be seen in Figure 18. It was found that the peak in the retention time between 14.74-14.99 min (compound no.1) has the antiviral activity against HCV. From the mass analysis, it was known that the mass of the compound is 604.3978 Da. Using the data of the compound mass and the UV spectrum as well as submitting it in the Dictionary Natural Product, it was then figured out that compound no. 1 is Bafilomycin D. In addition, by comparing with the pure Bafilomycin D, it was also revealed that the compound no. 1 is Bafilomycin D (Figure 19).

3.5 Taxonomic study of strain 196526CR 3.5.1 Morphology and melanin production Strain 196526CR could be grown well on ISP 2, ISP 5, ISP 6, ISP 7, SSM+T and SSM-T medium. Growth in ISP 3 was sparse and no growth was observed in ISP 4. The strain produced no melanin in the melanin production medium (Table 24). The morphology of the isolate 196526CR in GYM and ISP 2 medium can be seen in Figure 20.

3.5.2 16S rRNA gene analysis The almost complete 16S rRNA gene (1,458 nt) was used in the EzTaxon analysis (http://www. ezbiocloud.net/taxonomy)156. It was found that strain 196526CR was close to the type strains of Amycolatopsis thermalba SF45T (98.85%), Amycolatopsis deserti GY024T (98.84%), Amycolatopsis methanolica 239T (98.75%) and Amycolatopsis endophytica KLBMP 1221T (98.75%). In a phylogenetic tree, the isolate 196526CR formed a distinct subclade and clustered with Amycolatopsis deserti GY024T, close to Amycolatopsis endophytica KLBMP 1221T and Amycolatopsis thermophile GY088T by using the neighbour-Joining method. The subclade consisting of strain 196526CR and Amycolatopsis deserti GY024T were also founded in the tree constructed maximum-parsimony algorithm (Figure 21).

3 Results

Figure 18. LC-HRMS analysis result of raw extract of strain MAE 1-11 produced by SYP medium. A: Chromatogram of raw extract detected by 200-600 nm; B: UV Spectrum of compound no. 1 from the chromatogram; C: Mass spectrum of compound no. 1 from the chromatogram.

3 Results

Figure 19. LC-HRMS analysis result of Bafilomycin D. A: Structure molecule of bafilomycin D and its chromatogram detected by 200-600 nm and the; B: UV Spectrum of bafilomycin D; C: Mass spectrum of bafilomycin D.

3 Results

Table 24. Growth and characteristics of isolate 196526CR grown on various agar media after incubation for 14 days at 30°C

Agar medium Growth Substrate Aerial Soluble mycelium mycelium pigment color color Yeast extract-malt Good Signal yellow Pure white None extract (ISP 2) Oatmeal (ISP 3) Sparse Sand yellow Cream None Inorganic salt- None None None None starch (ISP 4) Glycerol- Good Sand yellow Pure white None asparagine (ISP 5) Peptone-yeast Good Honey yellow None None extract-iron (ISP 6) Tyrosine (ISP 7) Good Golden yellow Traffic white None Synthetically Suter Good Golden yellow Cream None medium with tyrosine (SSM+T) Synthetically Suter Good Golden yellow Light ivory None medium without tyrosine (SSM-T)

Figure 20. Morphology of strain 196526CR on GYM (left) and ISP 2 medium (right)

3 Results

Figure 21. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences between strain 196526CR and its closely related neighbours. The evolutionary history was inferred using the Neighbor-Joining method161. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches162. The evolutionary distances were computed using the Kimura 2-parameter method175. There were a total of 1344 positions in the final dataset. Evolutionary analyses were conducted in MEGA X158. Asterisks and filled circles indicate branches of the tree that were also found using the maximum-likelihood and maximum-parsimony tree-making algorithms, respctively.

3.5.3 Physiological and biochemical characteristic Strain 196526CR grew in the presence of 5% NaCl. The strain could also grow well by using glucose and fructose. Weak growth was detected on the medium containing arabinose, xylose, mannitol, and rhamnose. The results from API ZYM suggested that the strain possessed substantial enzymatic activities for β-Glucosidase and N-acetyl- beta-glucoseamidase. It also had good activities for phosphatase alkaline, phosphatase acid, urease, and gelatinase (Table 25).

3.5.4 Secondary metabolites produced by strain 196526CR In 5294HG-S medium, strain 196526CR produced coproporphyrin III, zinc coproporphyrin III, and nitrosoxacin C (Figure 22). Coproporphyrin III and zinc coproporphyrin III was detected both in the biomass and in the XAD extract, while nitrosoxacin C was found in the biomass extract (Figure 23 and Figure 24). It could

3 Results inhibit Bacillus substilis (MIC = 33.4 µg/ml) and Staphylococcus aureus (MIC = 33.4 µg/ml).

Table 25. Some physiology properties of strain 196526CR

Characteristic Observation Observation Use of carbohydrate Use of carbohydrate Glucose + Mannitol (+) Arabinose (+) Fructose + Sucrose - Rhamnose (+) Xylose (+) Raffinose - Inositol - Cellulose - NaCl tolerance 5% API ZYM API ZYM + Naphtol-AS-BI- - Phosphatase alkaline phosphohydrolase Esterase (C4) - α-Galactosidase - Esterase Lipase (C8) (+) β-Galactosidase (+) Lipase (C14) (+) β-Glucuronidase - Leucin arylamidase ++ α-Glucosidase - Valine arylamidase ++ β-Glucosidase ++ (+) N-acetyl-beta- ++ Cystine arylamidase glucoseamidase Trypsin (+) α-Mannosidase (+) Chymotrypsin - α-Fucosidase - Phosphatase acid + API Coryne API Coryne Nitrate reduction - Gelatine(hydrolysis) + Pyraziamidase - Glucose fermentation - Pyrrolidonyl - - Ribose fermentation arylamidase Alkaline phosphatase + Xylose fermentation - β-Glucuronidase - Mannitol fermentation - β-Galactosidase - Maltose fermentation - α-Glucosidase - Lactose fermentation - N-acetyl -beta + - Sucrose fermentation glucoseamidase Esculin (beta + - Glycogen fermentation glucosidase) Urease + ++ more positive result; + positive result; - negative result; (+) weakly positive result

3 Results

Figure 22. Structure of secondary metabolites produced by by strain 196526CR.

3 Results

Coproporphyrin Zn-Coproporphyrin III III

Zn-Coproporphyrin III

Coproporphyrin III UV and MS spectrum UV and MS spectrum

Figure 23. Chromatogram, UV and MS spectrum of Coproporphyrin III and Zn- Coproporphyrin III found in the XAD extract of strain 196526CR.

3 Results

Nitrosoxacin C

Nitrosoxacin C UV and MS spectrum

Figure 24. Chromatogram, UV and MS spectrum of Nitrosoxacin C found in the biomass extract of strain 196526CR.

3.6 Taxonomic study of Streptomyces sp. ASO4wet 3.6.1 Morphology and physiology After incubation for four weeks, strain ASO4wet formed aerial mycelium with no spore was detected on ISP3 agar (Figure 25). From the physiological study, it was found that strain ASO4wetT grew at 15-37oC (optimum at 25-30oC) and pH 6-9 (optimum at pH 7). Antibiotic susceptibility test suggested that isolate ASO4wet was sensitive to ampicillin (10 µg/disc), erythromycin (15 µg/disc), gentamycin 30 (µg/disc), penicillin G (6 µg/disc), tetracycline (30 µg/disc), vancomycin (30 µg/disc), and rifampicin (5 µg/disc); nevertheless, it was resistant to cefotaxime (30 µg/disc).

3 Results

Figure 25. Scanning electron micrographs of aerial mycelium with no spore detected of strain ASO4wetT after incubation on ISP 3 agar for 4 weeks at 30°C.

3.6.2 Chemotaxonomy The cell-wall of isolate ASO4wetT contained LL-diaminopimelic acid. Glucose and xylose were detected in its whole-cell hydrolysates (Figure 26). The identified fatty acids in strain ASO4wetT were iso-C16:0 (35.01%), anteiso-C15:0 (21.97%), iso- C15:0 (13.75%), anteiso-C17:0 (8.80%), and iso-C14:0 (6.31%) (Figure 27). Menaquinone MK-9(H8) and MK-9(H6) were detected in a ratio of 12:1 (Figure 28). The polar lipid compositions were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidyl-N-methyl- ethanolamine, phosphatidylinositol mannoside, and four unidentified polar lipids (Figure 29).

3.6.3 16S rRNA gene analysis By using BLAST or basic local alignment search tool from NCBI server (http:// www.ncbi.nlm.nih.gov/BLAST/)179, it was founded that isolate ASO4wetT belongs to the genus Streptomyces. The strain was closely related to Streptomyces karpasiensis K413T (98.87%), Streptomyces glycovorans YIM M 10366T (98.38%), and Streptomyces abyssalis M 10400T (97.53%). It was located in the clade together with Streptomyces karpasiensis K413T and Streptomyces glycovorans YIM M 10366T that was supported by 89% bootstrap value in the neighbour-joining tree based on the 16S rRNA gene sequence (Figure 30). The relationship between these strains in the phylogenetic tree was also retrieved by using maximum-likelihood and maximum- parsimony algorithm.

3 Results

3.6.4 Full genome analysis The draft genome of strain ASO4wetT was derived from PacBio-Sequencing. There was in total 7,377,472 bp was measured and the genome coverage was 117x. The genome was assessed by using Illumina MiSeq data and it encodes for about 6,500 genes after Prokka annotation. The G+C content was found to be 70.24 mol%.

Figure 26. TLC chromatogram of whole-cell sugar analysis (left) and analysis amino acid of the cell wall (right) of isolate ASO4wet. S1: Standard 1; S2: Standard 2; X: Xylose; A: Arabinose; Gal: Galactose; DPM: Diaminopimelic acid standard; LL-: LL- Diaminopimelic acid; Meso: meso-Diaminopimelic acid; ASO4: isolate ASO4wet.

3 Results

RT Response Ar/H RFact ECL Peak Name Percent 1.629 3.696E+8 0.026t ---- 7.010 SOLVENT PEAK ---- 2.536 137 0.023 ---- 8.810 ---- 4.385 334 0.027 1.064 11.608 12:0 ISO 0.08 5.448 830 0.036 1.020 12.612 13:0 ISO 0.20 5.551 906 0.033 1.017 12.701 13:0 ANTEISO 0.22 6.738 26965 0.035 0.986 13.617 14:0 ISO 6.31 7.259 863 0.037 0.976 13.998 14:0 0.20 8.219 60288 0.038 0.962 14.622 15:0 ISO 13.75 8.360 96480 0.039 0.960 14.714 15:0 ANTEISO 21.97 8.581 628 0.035 0.958 14.857 15:1 B 0.14 8.799 2495 0.040 0.955 14.999 15:0 0.56 9.565 21520 0.042 0.948 15.459 16:1 ISO H 4.84 9.847 156126 0.042 0.946 15.628 16:0 ISO 35.01 10.162 1862 0.040 0.943 15.817 16:1 CIS 9 0.42 10.463 9394 0.043 0.941 15.998 16:0 2.10 11.187 933 0.042 0.937 16.417 16:0 9? METHYL 0.21 11.368 4876 0.044 0.937 16.521 17:1 ANTEISO C 1.08 11.556 14143 0.043 0.936 16.629 17:0 ISO 3.14 11.716 39661 0.045 0.935 16.722 17:0 ANTEISO 8.80 12.006 1619 0.047 0.934 16.889 17:0 CYCLO 0.36 12.194 847 0.044 0.934 16.998 17:0 0.19 12.456 956 0.046 0.933 17.147 16:0 ISO 3OH 0.21 13.310 1004 0.041 0.932 17.632 18:0 ISO 0.22

Figure 27. GC chromatogram of fatty acid analysis of isolate ASO4wet.

3 Results

Figure 28. Menaquinones detected in isolate ASO4wet. A: Chromatogram from LC-MS of detected menaquinones; B: structure of menaquinone-9 (MK-9); C: UV Spectrum of menaquinone.

Figure 29. Polar lipid observed in strain ASO4wet. DPG: diphosphatidylglycerol; PE: phosphatidethanolamine; PL: unknown phospholipid; GL1-3: unknown glycolipid; PGL: unknown phosphoglycolipid; PIM: phosphatidylinositol mannoside.

3 Results

Figure 30. Neighbour-joining tree based on 16S rRNA gene sequences (1408 positions in the final dataset) showing relationships between Streptomyces sp. ASO4wetT and its closely related Streptomyces species. The evolutionary distances were determined by using the Kimura 2-parameter method175. Asterisks mean branches of the tree that were also found using the maximum-likelihood and maximum-parsimony tree-making algorithms. ML and MP specify nodes that were also recovered using the maximum- likelihood and maximum-parsimony respectively. Numbers at the nodes are percentage bootstrap values based on a neighbour-joining analysis of 1,000 replicates, only values above 50 % are shown. Bar 0.010 substitutions per nucleotide position.

4 Discussion

4 Discussion 4.1 Isolated Actinobacteria and their bioactivity In the isolation of Actinobacteria, the soil samples were treated by heating at 60°C for 30 minutes before they were applied to the selection medium. Many vegetative bacterial cells are killed at 60°C. It is known that many vegetative yeast cells are more heat-sensitive than the vegetative bacterial cells and it is also previously reported that typically Gram-positive bacteria are more heat resistant than Gram-negative bacteria180. Some Actinobacteria living in soil, such as Actinomyces, Streptomyces, and Micromonospora form spores that can resist heat stress. Streptomyces, for example, are able to produce numerous hydrophobic spores that are moderately dense coat, contain small protective molecules like some group of sugars (trehalose), and consist of heat shock proteins181.

The selection medium (5336 medium) for Actinobacteria isolation contains starch and casein. The combination of starch as the carbon source and casein as the nitrogen source was reported previously as one of the best media that permits good growth for Streptomyces. Moreover, it is also known that most of Streptomyces strains can use starch as the carbon source182. The medium also comprises magnesium sulfate

(MgSO4), which is essential for the cell division of the bacteria. Magnesium ion is more needed for Gram-positive bacteria than for Gram-negative bacteria. This probably because Gram-positive bacteria incorporate the magnesium ion into the complex structure of their cell wall183.

Nalidixic acid, an antibacterial compound, is also one of the components in the selection medium. It inhibits DNA replication in some bacteria, such as Escherichia coli and Bacillus substilis184. It is known as a selective, immediate, and reversible inhibitor of bacterial DNA synthesis185. The other antimicrobial compound used in the selection medium is cycloheximide. Cycloheximide is a glutarimide derivative that has property as an antifungal agent by inhibiting protein synthesis. The compound binds the ribosome and blocks the elongation phase of translation process186,187. Some studies have previously reported the usage of these antimicrobial agents in the selection media for some Actinobacteria isolation188,189.

In this study, plenty of Actinobacteria were isolated by using the preheated method and 5336 medium. The contamination from fungi and other bacteria could also be reduced with the addition of antifungal and antibacterial agents. Many of the isolated

4 Discussion

Actinobacteria in this investigation belongs to Streptomyces. About 70% of the characterized strains were identified as Streptomyces and from the identified Streptomyces, only 27% of them had less than 99.50% similarity from the type strains based on 16S rRNA gene analysis. One of those is isolate SHP 1-2 that had 99.03% similarity to Streptomyces viridochromogenes NBRC 3113T. The extract produced by strain SHP 1-2 from 5294 medium could inhibit moderately the growth of Staphylococcus aureus and strongly inhibit the growth of Bacillus substilis and Micrococcus luteus.

For other strains that were identified closely to non-Streptomyces type strains according to the 16S rRNA analysis, there are 24% of them, which had less than 99.5% similarity from the type strain. One of the isolated non-Streptomyces strain is isolate 196526CR, which had 98.85% similarity to Amycolatopsis thermalba SF45T (98.85%). It produced an extract from 5294 medium that could inhibit, although moderately, the growth of Bacillus subtilis, Staphylococcus aureus, Mucor hiemalis, and Candida albicans. From the isolated strains that can yield extracts with moderate and strong activity against some tested microbes, almost 70% of them are Streptomyces strains.

Ten isolated strains showed strong activity against hepatitis C virus (HCV). Three of them were closely related to Kitasatopora species according to the result from EzBioCloud server (https://www.ezbiocloud.net)156. These strains are isolate DHE 2-1, MAE 1-11, and C190221. Kitasatospora and Streptomyces are very closely related because both of them belong to the same family Streptomycetaceae. They also have a similar lifestyle and morphology. In the capability of producing bioactive compounds, Kitasatopora could also be equivalent to Streptomyces. The difference between these two genera is on their cell-wall peptidoglycan composition. The cell- wall peptidoglycan of Streptomyces comprises LL-diaminopimelic acid (DAP), whereas Kitasatospora contains not only LL-diaminopimelic acid (LL-DAP) but also meso-DAP190.

4.2 Description of strain SHP 1-2 and its secondary metabolites Strain SHP 1-2 was isolated from soil collected in Enggano Island, Indonesia. The isolate has a broadly branched substrate mycelium that bears aerial hyphae which develop into spiral chains of smooth-surfaced spores on ISP 3 agar medium. It is a mesophilic, halotolerant, and non-melanin producing bacterium. The 16S rRNA gene

4 Discussion analysis suggested that isolate SHP 1-2 is close related to Streptomyces species. It has high G+C content in its genomic DNA. The morphologic and chemotaxonomic characteristics of strain SHP 1-2 have corresponding profiles to some other Streptomyces species that have been previously reported 191–193. Therefore, it is determined that strain SHP 1-2 belongs to Streptomyces species.

In the phenotypic characteristic, many differences are found between strain SHP 1-2 and the closest relative strains based on 16S rRNA gene sequence, i.e., Streptomyces malachitofuscus DSM 40332T (99.03%), Streptomyces viridochromogenes DSM 40110T (99.03%), and Streptomyces misionensis DSM 40306T (98.96%), as well as the closest one from the MLSA distance (Streptomyces fumigatiscleroticus DSM 43154T, MLSA distance: 0.058). The significant difference is in the basis of single carbon source usage, as for strain SHP 1-2, it can only use glucose, while the others are able to use several of sole carbon sources. Furthermore, based on the enzymatic system, strain SHP 1-2 shows a substantial distinction with the others. The chymotrypsin activity was not detected in strain SHP 1-2, while in the others, the chymotrypsin activities are observed. It can be inferred; therefore, that strain SHP 1- 2, in the phenotypic features, is different from the compared type strains.

Recently, however, there are some problems to delineate Streptomyces strains. This because of the vast amounts of isolates and insufficiently determined species, the weak resolution of 16S rRNA gene sequence as the phylogenetic standard, complexities in comparing essential phenotypic features between the strains, and inconvenience of DNA–DNA hybridization (DDH) and DNA fingerprinting for rapid analysis. Multilocus sequence analysis (MLSA) has been used recently in microbial systematic. MLSA has been recently used for delineation of Streptomyces species because of its robustness in the molecular method130.

By using five house-keeping genes, i.e., atpD, gyrB, recA, rpoB and trpB in MLSA analysis, it was found that strain SHP 1-2 showed to have MLSA distance more than 0.007 with all of the other related species. The MLSA distance of 0.007 is equivalent to 70 % DNA–DNA homology130. The MLSA distance that is more than 0.007 means that the strain can be separated with the other compared strains. After studying polyphasic taxonomy, that includes the genotypic and phenotypic comparison between strain SHP 1-2 and the close strains, it can be concluded that the strain SHP 1-2 is a novel species in the Streptomyces genus.

4 Discussion

Piericidin A1 is the most active compound produced by strain SHP 1-2. This compound is also produced by other Streptomyces species such as Streptomyces mobaraensis and Streptomyces pactum194. The mechanism of the activity against some tested organisms is suggested due to its ability to inhibit mitochondrial and bacterial NADH-ubiquinone oxidoreductases195. It was also previously reported that the compound possesses the antibacterial activity against Gram-negative bacteria Chromobacterium violaceum CV026 by inhibiting the quorum-sensing mechanism196.

Two new molecules that were isolated from strain SHP 1-2 are indolactam derivatives. These compounds have a moderate activity to inhibit the proliferation of KB-3-1 and PC-3 cells lines. As for indolactam variant II, it also has modest activity against SK- OV-3 cell line. The other indolactam structure, indolactam V, was also isolated from strain SHP 1-2. It has been reported previously that indolactam V has numerous biological activities. It can stimulate protein kinase C (PKC), generate pancreatic progenitor cells from human embryonic stem cells (hESCs), and induce the Epstein- Barr virus early antigen (EBV-EA) of Raji cells (B lymphocyte)197–199. This compound was previously also isolated from Streptomyces blastmyceticum NA3417 and known as a biosynthetic intermediate of teleocidins, which are identified as influential skin tumor promoters200.

Two other compounds produced by strain SHP 1-2 are cyclo(phenylalanyl-prolyl) and lyngbyatoxin A. Cyclo(phenylalanyl-prolyl) is diketopiperazine molecule that was also isolated previously from Streptomyces sp. (NPS008187)201. The other study suggested that cyclo(phenylalanyl-prolyl) could be produced by Lactobacillus plantarum strain (MiLAB 393). The compound was also reported having biological activity property as antifungal agent202. As for lyngbyatoxin A, this compound was first reported in the previous study to be found in marine cyanobacterium Moorea producens (formerly Lyngbya majuscula). It is the causative compound of seaweed dermatitis. It has a highly inflammatory effect, vigorous tumor-promoting activity and capability to stimulate protein kinase C isozymes. The compound was determined to be the same as teleocidin A-1, which was isolated from Streptomyces mediocidicus203.

4.3 Description of strain MAE 1-11 and its secondary metabolites The calculation from EzBioCloud server (https://www.ezbiocloud.net)156 using 16S rRNA gene suggested that strain MAE-11 was very close to the type

4 Discussion strain Kitasatospora albolonga NBRC 13465T (99.93%). The phylogenetic tree analysis result, however, indicated that strain MAE-11 was closer to Streptomyces cavourensis NBRC 13026T than to K. albolonga NBRC 13465T according to the neighbour-joining algorithm. The clade was also supported by a relatively high percentage of bootstrap value.

Kitasatospora is the genus of Actinobacteria, which was firstly proposed in 1982 and the name itself refers to Kitasato, who was a Japanese bacteriologist98. Kitasatospora and Streptomyces are very closely related in the taxonomy as both of them are in the same family Streptomycetaceae and also look similar in their morphology. The thing that makes them different is the cell-wall composition. Streptomyces contains LL- diaminopimelic acid and no meso-diaminopimelic acid, whereas Kitasatopora has both of the LL- and meso-diaminopimelic acid204. The other thing which different is that Kitasatopora has galactose as the diagnostic sugar, while Streptomyces has no diagnostic sugar125.

Strain MAE 1-11 was isolated from soil collected in the mangrove area of Enggano Island, Indonesia. It is a halotolerant and melanin producer bacterium. Strain MAE 1- 11 can be distinguished from K. albolonga NRRL B-3604T (= NBRC 13465T) on the basis of the enzymatic system. K. albolonga NRRL B-3604T (= NBRC 13465T) does not have activity for phosphatase alkaline, esterase (C4), alpha-chymotrypsin, and alpha-glucosidase, while strain MAE 1-11 have all these enzymes205,206. Strain MAE 1-11 is also different from S. cavourensis NBRC 13026T according to the enzymatic activity. Some enzymes were not detected in S. cavourensis NBRC 13026T, such as esterase (C8), trypsin, phosphatase acid, naphtol-AS-BI-phosphohydrolase, and beta- galactosidase206, whereas isolate MAE 1-11 equipped with these enzymes.

The primary compound produced by strain MAE 1-11 is bafilomycin D. The compound has antiviral activity against HCV. Bafilomycin D is a 16-membered ring macrolide antibiotic that possesses activity in the inhibition of V- and P-ATPases, although it is not as strong as bafilomycin A1. V-ATPases are vacuolar-type, proton- translocating ATPases (V-ATPases), while P-ATPases are ATPases with phosphorylated states (P-ATPases). Both of them can be found in animal and plant cells, as well as in yeast, fungi, and bacteria207. Bafilomycin D was also previously isolated from Streptomyces griseus Tü 2599, Streptomyces sp. YIM56209,

4 Discussion

Streptomyces albolongus (novel name is Kitasatospora albolonga) strain YIM 101047208–210.

The mechanism of antiviral bafilomycin D against HCV may be similar to how bafilomycin A1 can inhibit influenza A virus (IAV) replication. Bafilomycin A1 at relatively high concentration (≥10 nM) was reported that it could inhibit V-ATPase and decrease endosome acidification as well as lysosome number; consequently, it diminished IAV replication. However, it could also trigger cytotoxicity for the host cell. Interestingly, at a shallow concentration (0.1 nM), this molecule was still able to inhibit the replication, and the release of IAV albeit this amount of bafilomycin A1 is neither causing reduction of lysosome number nor toxic to the host cell211.

4.4 Description of strain 196526CR and its secondary metabolites The 16S rRNA gene analysis, according to the result from EzBioCloud server (https://www.ezbiocloud.net)156, suggested that strain 196526CR was very close to the type strain Amycolatopsis thermalba SF45T (98.85%), however, based on the constructed phylogenetic tree, Amycolatopsis deserti GY024T was the closest strain to strain 196526CR. The clade formed in the phylogenetic tree between strain 196526CR and A. deserti GY024T was found both with the neighbour-joining and maximum- likelihood method, although in neighbor-joining method, this clade was only supported by less than 50% of bootstrap values.

Amycolatopsis is one of the genera in Actinobacteria and belongs to the family Pseudonocardiaceae212. Until now, the genus Amycolatopsis includes already 76 published species (http://www.bacterio.net/amycolatopsis.html). Amycolatopsis can be distinguished from the other genera in the family Pseudonocardiaceae by using genotypic and phenotypic assessment. It has no sporangia; no motile spores; the diagnostic sugars are arabinose and galactose; the phospholipid system contains PE (phosphatidylethanolamine), DPG (diphosphatidylglycerol), PG (phosphatidylglycerol), and PI (phosphatidylinositol; and the predominant menaquinone is MK- 9(H4)213. The spores in Amycolatopis can be found both in aerial and substrate hyphae. The spores in aerial hyphae are formed in chains, the genus also has fragmented substrate hyphae214 and contains meso-diaminopimelic acid in its cell-wall structure212. The genus-specific primers for Amycolatopsis were also constructed in the previous study for detecting Amycolatopsis in soil samples215.

4 Discussion

Some antibiotics are produced by genus Amycolatopsis. Species Amycolatopsis orientalis is the producer of vancomycin, which is a strong glycopeptide antibiotic that has activity against methicillin-resistant Staphylococcus aureus (MRSA) infections216. Balhimycin, which is categorized to the vancomycin class of glycopeptide antibiotic, is produced by Amycolatopsis Amycolatopsis balhimycina. Balhimycin was found to be correspondingly strong as vancomycin against MRSA strains and more powerful than vancomycin, specifically against Clostridium strains217. Amycolatopsis mediterranei is well-known as a producer of Rifamycin B, which is used for the treatment of tuberculosis and other diseases caused by Mycobacterium. Rifamycin B belongs to the ansamycin family, which is characterized by a macrocyclic structure containing aromatic moiety bridged by an aliphatic ansa chain218,219.

Strain 196526CR was isolated from soil in Bali, Indonesia. The strain is a halotolerant and non-melanin producing bacterium. The isolate can grow well by using glucose and fructose as a single carbon source, however, there was no alpha-glucosidase activity detected in this strain. Alpha-glucosidase is one of the essential enzymes in starch hydrolysis to produce glucose220. This may be the reason why the strain can not grow in ISP 4 medium, which contains only starch as the single source of carbon.

Not like strain 196526CR, one of the close strains, A. deserti GY024T, have no enzymatic activity in beta-glucosidase. Another close type strain, A. thermalba SF45T were found not having N-acetyl-beta-glucoseamidase and acid phosphatase221, whereas, for the strain 196526CR, these enzymes are produced. This finding may suggest that strain 196526CR can probably be distinguished from A. deserti GY024T and A. thermalba SF45T based on the enzymatic system.

Some compounds that are produced by strain 196526CR are coproporphyrin III, zinc coproporphyrin III, and nitrosoxacin C. Coproporphyrin III is a tetrapyrrole moiety and is the primary intermediate of heme biosynthesis in Amycolatopsis and other Actinobacteria. Heme is very important in the numerous biological process. It functions as a prosthetic group for various proteins, such as cytochromes, globins, catalases, peroxidases, and transporters. These proteins are essential in respiration, photosynthesis, metabolism, and transport of oxygen in organisms222,223.

Zinc coproporphyrin III is the derivative of coproporphyrin III which is bound to zinc metal. The previous study suggested that coproporphyrin III showed a higher affinity

4 Discussion for zinc than other metals224. The compound was also found in Streptomyces sp. AC8007 and has some biological activities, such as a histamine-release inhibitor and a potent photosensitizer for controlling tumor growth in photodynamic cancer therapy225.

Nitrosoxacin C was isolated by using bioassay-guided isolation against Staphylococcus aureus since the molecule was difficult to be detected by the UV detector. The MIC results suggest that Nitrosoxacin C has moderate antimicrobial activity against Staphylococcus aureus and Bacillus substilis. It is not known previously that Nitrosoxacin C has antibacterial activity against these bacteria. The previous study reported that Nitrosoxacin C was isolated from Streptomyces sp. AA4091 and it is known to have the activity as a 5-lipoxygenase inhibitor. The possible mechanism of 5-lipoxygenase inhibitory activity is because of its chelating activity226. 5-lipoxygenase inhibitors are used in the treatment of inflammatory bowel disease (IBD)227.

4.5 Description of strain ASO4wet Strain ASO4wet was isolated from the sponge in a deep-sea collected from the North Atlantic Ocean. Some of the characterizations of this strain were conducted previously by Landwehr147. These include 16S rDNA analysis, morphology and melanin production, some physiological studies, MALDI-TOF analysis by using ribosomal protein, DNA-DNA hybridization, and ribotyping analysis with some closely related strains. This recent study reports the other characteristics such as morphology by scanning electron microscopy (SEM), some physiology studies, chemotaxonomy assessment, phylogenetic tree construction based on 16S rRNA gene, and full genome sequencing for determining the size of the genome as well as the G+C content.

Strain ASO4wet is an aerobic and Gram-positive bacterium that forms branched substrate mycelium with the aerial hyphae that can be seen in ISP3 and ISP7 agar medium. The strain grows well on ISP2, ISP3, ISP4, and ISP5 after two weeks incubation at 30°C. The enzymatic system comprises esterase (C4), trypsin, chymotrypsin, β-galactosidase, and α-glucosidase. The NaCl tolerance of this strain is 0-10% (w/v) NaCl. The strain can grow with glucose, arabinose, sucrose, xylose, inositol, mannitol, fructose, rhamnose, and raffinose as the sole carbon sources147.

4 Discussion

Spores were not observed in ISP3 even after four weeks incubation at 30°C. The optimum growth was detected at 25–30°C and at pH 7. From the 16S rRNA and chemotaxonomy analysis results as well as the high G+C content in its genomic DNA, it is determined that strain ASO4wet is a member of the genus Streptomyces. Strain ASO4wetT formed a stable subclade with Streptomyces karpasiensis K413T with high bootstrap value and adjacent to Streptomyces glycovorans YIM M 10366T. The position in the phylogenetic tree between these Streptomyces strains was also confirmed by the maximum-likelihood and maximum-parsimony methods.

Strain ASO4wet can be separated from its closest strains based on the enzymatic system such as lipase (C14), beta-galactosidase, beta-glucosidase, the use of single carbon, MALDI-TOF, and ribotyping analysis. Furthernore, the DNA-DNA hybridization results with the closest Streptomyces type strains are lower than 70%147. Therefore, it is suggested that strain ASO4wet represents a novel species within the genus Streptomyces.

5 Summary

5 Summary Hundreds of Actinobacteria were successfully isolated from Indonesian source samples. However, due to the limitation of time allocation, only around half of them were characterized. The study was focused on strain SHP 1-2, MAE 1-11, and 19626CR due to its ability to produce active extracts against either some microbes or hepatitis C virus (HCV). Another strain that was used in this study was strain ASO4wet that was isolated previously from deep-sea in the North Atlantic Ocean. Strain SHP 1-2 was known to produce two novel indolactam derivatives. It was also suggested that strain SHP 1-2 is the novel species in the genus Streptomyces. Bafilomycin D, which was produced by strain MAE -11, was revealed to have antiviral properties against HCV. This strain is closely related to Kitasatopora and Streptomyces species. Strain 196526CR, which was characterized as Amycolatopsis species, generated nitrosoxacin C that has antimicrobial property. The antimicrobial activity of nitrosoxacin C was firstly reported in this study. A chemotaxonomic study of strain ASO4wet gave supporting data that this strain belongs to the genus Streptomyces.

Streptomyces species can typically be detected by their aerial mycelium formation. However, some non-Streptomyces genera such as Kitasatospora, Amycolatopsis, and Pseudonocardiopsis also have similar aerial mycelium as Streptomyces. The chemotaxonomic study, especially for amino acid of cell-wall analysis, can distinguish between Streptomyces and non-Streptomyces species based on the present of LL- diaminopimelic acid and its isomer. The identification based on 16S rRNA sequence further is needed to clarify which type strain the isolates are close to. Around half of the isolates were not studied yet either for taxonomic characterization or for metabolite production. Further study is needed for clarifying the species and its secondary metabolites.

6 References

1. Katz L, and Baltz RH. Natural product discovery: past, present, and future. J Ind Microbiol Biotechnol. 2016;43(2-3):155-176. doi:10.1007/s10295-015- 1723-5 2. Demain AL. Importance of microbial natural products and the need to revitalize their discovery. J Ind Microbiol Biotechnol. 2014;41(2):185-201. doi:10.1007/s10295-013-1325-z 3. Ding T, Yang L-J, Zhang W-D, and Shen Y-H. The secondary metabolites of rare actinomycetes: chemistry and bioactivity. RSC Adv. 2019;9(38):21964- 21988. doi:10.1039/C9RA03579F 4. Agrawal S, Acharya D, Adholeya A, Barrow CJ, and Deshmukh SK. Nonribosomal peptides from marine microbes and their antimicrobial and anticancer potential. Front Pharmacol. 2017;8(NOV):1-26. doi:10.3389/fphar.2017.00828 5. Čihák M, Kameník Z, Šmídová K, Bergman N, Benada O, Kofroňová O, Petříčková K, and Bobek J. Secondary metabolites produced during the germination of Streptomyces coelicolor. Front Microbiol. 2017;8(December):1-13. doi:10.3389/fmicb.2017.02495 6. Demain AL, and Sanchez S. Microbial drug discovery: 80 Years of progress. J Antibiot (Tokyo). 2009;62(1):5-16. doi:10.1038/ja.2008.16 7. Singh R, Kumar M, Mittal A, and Mehta PK. Microbial metabolites in nutrition, healthcare and agriculture. 3 Biotech. 2017;7(1):15. doi:10.1007/s13205-016-0586-4 8. Bérdy J. Bioactive microbial metabolites. J Antibiot (Tokyo). 2005;58(1):1-26. doi:10.1038/ja.2005.1 9. Fleming A. On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae. Br J Exp Pathol. 1929;10:226-236. 10. Waksman SA, and Tishler M. Chemical nature of actinomycin. J Biol Chem. 1942;142:519-528. 11. Labeda DP. Transfer of the type strain of Streptomyces erythraeus (Waksman 1923) Waksman and Henrici 1948 to the genus Saccharopolyspora Lacey and Goodfellow 1975 as Saccharopolyspora erythraea sp. nov., and designation of a neotype strain for <. Int J Syst Bacteriol. 1987;37(1):19-22. doi:10.1099/00207713-37-1-19 12. Fischbach MA, and Walsh CT. Antibiotics for emerging pathogens. Science (80- ). 2009;325(5944):1089-1093. doi:10.1126/science.1176667 13. Fischer C, Lipata F, and Rohr J. The complete gene cluster of the antitumor agent gilvocarcin V and its implication for the biosynthesis of the gilvocarcins. J Am Chem Soc. 2003;125(26):7818-7819.

6 References

doi:10.1021/ja034781q 14. Müller R, and Wink J. Future potential for anti-infectives from bacteria - How to exploit biodiversity and genomic potential. Int J Med Microbiol. 2014;304(1):3-13. doi:10.1016/j.ijmm.2013.09.004 15. Wohlleben W, Mast Y, Stegmann E, and Ziemert N. Antibiotic drug discovery. Microb Biotechnol. 2016;9(5):541-548. doi:10.1111/1751- 7915.12388 16. Risdian C, Primahana G, Mozef T, Dewi RT, Ratnakomala S, Lisdiyanti P, and Wink J. Screening of antimicrobial producing Actinobacteria from Enggano Island, Indonesia. AIP Conf Proc. 2018;2024(1):020039. doi:10.1063/1.5064325 17. Bode HB, Bethe B, Höfs R, and Zeeck A. Big effects from small changes: Possible ways to explore nature’s chemical diversity. ChemBioChem. 2002;3(7):619. doi:10.1002/1439-7633(20020703)3:7<619::AID- CBIC619>3.0.CO;2-9 18. Pettit RK. Small-molecule elicitation of microbial secondary metabolites. Microb Biotechnol. 2011;4(4):471-478. doi:10.1111/j.1751- 7915.2010.00196.x 19. Abdelmohsen UR, Grkovic T, Balasubramanian S, Kamel MS, Quinn RJ, and Hentschel U. Elicitation of secondary metabolism in actinomycetes. Biotechnol Adv. 2015;33(6):798-811. doi:10.1016/j.biotechadv.2015.06.003 20. Trivella DBB, and de Felicio R. The tripod for bacterial natural product discovery: Genome mining, silent pathway induction, and mass spectrometry- based molecular networking. mSystems. 2018;3(2):1-5. doi:10.1128/mSystems.00160-17 21. Ward A, and Allenby N. Genome mining for the search and discovery of bioactive compounds: The Streptomyces paradigm. FEMS Microbiol Lett. 2018;365(24):1-20. doi:10.1093/femsle/fny240 22. Barka EA, Vatsa P, Sanchez L, Gaveau-Vaillant N, Jacquard C, Klenk H-P, Clément C, Ouhdouch Y, and van Wezel GP. Taxonomy, physiology, and natural products of Actinobacteria. Microbiol Mol Biol Rev. 2016;80(1):1-43. doi:10.1128/MMBR.00019-15 23. Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald GF, Chater KF, and van Sinderen D. Genomics of Actinobacteria: Tracing the evolutionary history of an ancient phylum. Microbiol Mol Biol Rev. 2007;71(3):495-548. doi:10.1128/MMBR.00005-07 24. Labeda DP, Doroghazi JR, Ju KS, and Metcalf WW. Taxonomic evaluation of Streptomyces albus and related species using multilocus sequence analysis and proposals to emend the description of Streptomyces albus and describe Streptomyces pathocidini sp. nov. Int J Syst Evol Microbiol. 2014;64(PART 3):894-900. doi:10.1099/ijs.0.058107-0 25. Seipke RF, Kaltenpoth M, and Hutchings MI. Streptomyces as symbionts: An emerging and widespread theme? FEMS Microbiol Rev. 2012;36(4):862-876. doi:10.1111/j.1574-6976.2011.00313.x

6 References

26. Manteca A, Alvarez R, Salazar N, Yague P, and Sanchez J. Mycelium differentiation and antibiotic production in submerged cultures of Streptomyces coelicolor. Appl Environ Microbiol. 2008;74(12):3877-3886. doi:10.1128/AEM.02715-07 27. de Lima Procópio RE, da Silva IR, Martins MK, de Azevedo JL, and de Araújo JM. Antibiotics produced by Streptomyces. Brazilian J Infect Dis. 2012;16(5):466-471. doi:10.1016/j.bjid.2012.08.014 28. Terfehr D, Dahlmann TA, and Kück U. Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs. BMC Genomics. 2017;18(1):272. doi:10.1186/s12864-017-3663-0 29. Schimana J, Gebhardt K, Höltzel A, Schmid DG, Süssmuth R, Müller J, Pukall R, and Fiedler H-P. Arylomycins A and B, new biaryl-bridged lipopeptide antibiotics produced by Streptomyces sp. Tü 6075. I. Taxonomy, fermentation, isolation and biological Activities. J Antibiot (Tokyo). 2002;55(6):565-570. doi:10.7164/antibiotics.55.565 30. Smith PA, and Romesberg FE. Mechanism of action of the arylomycin antibiotics and effects of signal peptidase I inhibition. Antimicrob Agents Chemother. 2012;56(10):5054-5060. doi:10.1128/AAC.00785-12 31. Skinner RH, and Cundliffe E. Resistance to the antibiotics viomycin and capreomycin in the Streptomyces species which produce them. Microbiology. 1980;120(1):95-104. doi:10.1099/00221287-120-1-95 32. Aharonowitz Y, and Demain AL. Carbon catabolite regulation of cephalosporin production in Streptomyces clavuligerus. Antimicrob Agents Chemother. 1978;14(2):159-164. doi:10.1128/AAC.14.2.159 33. Yotsuji A, Mitsuyama J, Hori R, Yasuda T, Saikawa I, Inoue M, and Mitsuhashi S. Mechanism of action of cephalosporins and resistance caused by decreased affinity for penicillin-binding proteins in Bacteroides fragilis. Antimicrob Agents Chemother. 1988;32(12):1848-1853. doi:10.1128/AAC.32.12.1848 34. Jardetzky O. Studies on the mechanism of action of chloramphenicol. I. The conformation of chlioramphenicol in solution. J Biol Chem. 1963;238:2498- 508. doi:10.1016/0006-2952(85)90023-1 35. Piraee M, Magarvey N, He J, and Vining LC. The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular non-ribosomal peptide synthetase gene. Microbiology. 2001;147(10):2817-2829. doi:10.1099/00221287-147-10-2817 36. Svensson M-L, and Gatenbeck S. The presence of two serine racemases in Streptomyces garyphalus, a d-cycloserine producer. Arch Microbiol. 1981;129(3):213-215. doi:10.1007/BF00425253 37. Batson S, de Chiara C, Majce V, Lloyd AJ, Gobec S, Rea D, Fülöp V, Thoroughgood CW, Simmons KJ, Dowson CG, Fishwick CWG, de Carvalho LPS, and Roper DI. Inhibition of D-Ala:D-Ala ligase through a

6 References

phosphorylated form of the antibiotic D-cycloserine. Nat Commun. 2017;8(1):1939. doi:10.1038/s41467-017-02118-7 38. Steenbergen JN, Alder J, Thorne GM, and Tally FP. Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections. J Antimicrob Chemother. 2005;55(3):283-288. doi:10.1093/jac/dkh546 39. Rogers TO, and Birnbaum J. Biosynthesis of Fosfomycin by Streptomyces fradiae. Antimicrob Agents Chemother. 1974;5(2):121-132. doi:10.1128/AAC.5.2.121 40. Gao W, Wu Z, Sun J, Ni X, and Xia H. Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering. Virolle M-J, ed. PLoS One. 2017;12(7):e0181971. doi:10.1371/journal.pone.0181971 41. Spížek J, and Řezanka T. Lincomycin, clindamycin and their applications. Appl Microbiol Biotechnol. 2004;64(4):455-464. doi:10.1007/s00253-003- 1545-7 42. Fourmy D, Recht MI, and Puglisi JD. Binding of neomycin-class aminoglycoside antibiotics to the A-site of 16S rRNA. J Mol Biol. 1998;277(2):347-362. doi:10.1006/jmbi.1997.1552 43. Dulmage HT. The production of neomycin by Streptomyces fradiae in synthetic media. Appl Microbiol. 1953;1(2):103–106. 44. Flinspach K, Ruckert C, Kalinowski J, Heide L, and Apel AK. Draft genome sequence of Streptomyces niveus NCIMB 11891, producer of the aminocoumarin antibiotic novobiocin. Genome Announc. 2014;2(1):9140. doi:10.1128/genomeA.01146-13 45. Sugino A, Higgins NP, Brown PO, Peebles CL, and Cozzarelli NR. Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc Natl Acad Sci. 1978;75(10):4838-4842. doi:10.1073/pnas.75.10.4838 46. Vilches C, Mendez C, Hardisson C, and Salas JA. Biosynthesis of oleandomycin by Streptomyces antibioticus: Influence of nutritional conditions and development of resistance. J Gen Microbiol. 1990;136(8):1447-1454. doi:10.1099/00221287-136-8-1447 47. Fierro JF, Hardisson C, and Salas JA. Resistance to oleandomycin in Streptomyces antibioticus, the producer organism. Microbiology. 1987;133(7):1931-1939. doi:10.1099/00221287-133-7-1931 48. Falzone M, Crespo E, Jones K, et al. Nutritional control of antibiotic production by Streptomyces platensis MA7327: Importance of L-aspartic acid. J Antibiot (Tokyo). 2017;70(7):828-831. doi:10.1038/ja.2017.49 49. De Crécy-Lagard V, Blanc V, Gil P, Naudin L, Lorenzon S, Famechon A, Bamas-Jacques N, Crouzet J, and Thibaut D. Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: Molecular characterization of the first two structural peptide synthetase genes. J Bacteriol. 1997;179(3):705-713. doi:10.1128/jb.179.3.705-713.1997 50. Zachman-Brockmeyer TR, Thoden JB, and Holden HM. The structure of RbmB from Streptomyces ribosidificus, an aminotransferase involved in the

6 References

biosynthesis of ribostamycin. Protein Sci. 2017;26(9):1886-1892. doi:10.1002/pro.3221 51. Karray F, Darbon E, Oestreicher N, Dominguez H, Tuphile K, Gagnat J, Blondelet-Rouault MH, Gerbaud C, and Pernodet JL. Organization of the biosynthetic gene cluster for the macrolide antibiotic spiramycin in Streptomyces ambofaciens. Microbiology. 2007;153(12):4111-4122. doi:10.1099/mic.0.2007/009746-0 52. Luzzatto L, Apirion D, and Schlessinger D. Mechanism of action of streptomycin in E. coli: Interruption of the ribosome cycle at the initiation of protein synthesis. Proc Natl Acad Sci. 1968;60(3):873-880. doi:10.1073/pnas.60.3.873 53. Darken MA, Berenson H, Shirk RJ, and Sjolander NO. Production of Tetracycline by Streptomyces aureofaciens in Synthetic Media. Appl Microbiol. 1960;8(1):46-51. 54. Chopra I, and Roberts M. Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 2001;65(2):232–260. doi:10.1128/MMBR.65.2.232 55. Dzhavakhiya V, Savushkin V, Ovchinnikov A, Glagolev V, Savelyeva V, Popova E, Novak N, and Glagoleva E. Scaling up a virginiamycin production by a high-yield Streptomyces virginiae VKM Ac-2738D strain using adsorbing resin addition and fed-batch fermentation under controlled conditions. 3 Biotech. 2016;6(2):240. doi:10.1007/s13205-016-0566-8 56. Tiwari K, and Gupta RK. Rare actinomycetes: a potential storehouse for novel antibiotics. Crit Rev Biotechnol. 2012;32(2):108-132. doi:10.3109/07388551.2011.562482 57. Lazzarini A, Cavaletti L, Toppo G, and Marinelli F. Rare genera of actinomycetes as potential producers of new antibiotics. Antonie Van Leeuwenhoek. 2000;78(3-4):399-405. doi:10.1023/A:1010287600557 58. Vandamme P, Pot B, Gillis M, De Vos P, Kersters K, and Swings J. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev. 1996;60(2):407-438. 59. Ramasamy D, Mishra AK, Lagier J-C, Padhmanabhan R, Rossi M, Sentausa E, Raoult D, and Fournier P-E. A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species. Int J Syst Evol Microbiol. 2014;64(Pt 2):384-391. doi:10.1099/ijs.0.057091-0 60. Sadaka C, Ellsworth E, Hansen PR, Ewin R, Damborg P, and Watts JL. Review on abyssomicins: Inhibitors of the chorismate pathway and folate biosynthesis. Molecules. 2018;23(6):1-25. doi:10.3390/molecules23061371 61. Gomes KM, Duarte RS, and de Freire Bastos M do C. Lantibiotics produced by Actinobacteria and their potential applications (a review). Microbiology. 2017;163(2):109-121. doi:10.1099/mic.0.000397 62. Kim J, Shin D, Kim SH, Park W, Shin Y, Kim WK, Lee SK, Oh KB, Shin J, and Oh DC. Borrelidins C-E: New antibacterial macrolides from a saltern- derived halophilic Nocardiopsis sp. Mar Drugs. 2017;15(6):1-11. doi:10.3390/md15060166

6 References

63. Habibi D, Ogloff N, Jalili RB, Yost A, Weng AP, Ghahary A, and Ong CJ. Borrelidin, a small molecule nitrile-containing macrolide inhibitor of threonyl-tRNA synthetase, is a potent inducer of apoptosis in acute lymphoblastic leukemia. Invest New Drugs. 2012;30(4):1361-1370. doi:10.1007/s10637-011-9700-y 64. Hug JJ, Bader CD, Remškar M, Cirnski K, and Müller R. Concepts and methods to access novel antibiotics from actinomycetes. Antibiotics. 2018;7(2). doi:10.3390/antibiotics7020044 65. Braña AF, Sarmiento-Vizcaíno A, Pérez-Victoria I, Otero L, Fernández J, Palacios JJ, Martín J, De La Cruz M, Díaz C, Vicente F, Reyes F, García LA, and Blanco G. Branimycins B and C, antibiotics produced by the abyssal Actinobacterium Pseudonocardia carboxydivorans M-227. J Nat Prod. 2017;80(2):569-573. doi:10.1021/acs.jnatprod.6b01107 66. Nakamura G, Kobayashi K, Sakurai T, and Sono K. Cationomycin, a new polyether ionophore antibiotic produced by Actinomadura nov. sp. J Antibiot (Tokyo). 1981;34(11):1513-1514. doi:10.7164/antibiotics.34.1513 67. Herzog HL, Meseck E, Delorenzo S, Murawski A, Charney W, and Rosselet JP. Chemistry of antibiotics from Micromonospora. 3. Isolation and characterization of everninomicin D and B. Appl Microbiol. 1965;13(4):515- 520. 68. McNicholas PM, Najarian DJ, Mann PA, Hesk D, Hare RS, Shaw KJ, and Black TA. Evernimicin binds exclusively to the 50S ribosomal subunit and inhibits translation in cell-free systems derived from both Gram-positive and Gram-negative bacteria. Antimicrob Agents Chemother. 2000;44(5):1121- 1126. doi:10.1128/AAC.44.5.1121-1126.2000 69. Thornsberry C, Barry AL, Jones RN, Baker CN, Badal RE, and Packer RR. Antibacterial activity of fortimicin A compared with those of five other aminoglycosides, and factors affecting susceptibility tests. Antimicrob Agents Chemother. 1981;19(1):122-129. doi:10.1128/AAC.19.1.122 70. Moreau N, Jaxel C, and Le Goffic F. Comparison of fortimicins with other aminoglycosides and effects on bacterial ribosome and protein synthesis. Antimicrob Agents Chemother. 1984;26(6):857-862. doi:10.1128/AAC.26.6.857 71. Krause KM, Serio AW, Kane TR, and Connolly LE. Aminoglycosides: An overview. Cold Spring Harb Perspect Med. 2016;6(6):a027029. doi:10.1101/cshperspect.a027029 72. Phillips JW, Goetz MA, Smith SK, et al. Discovery of kibdelomycin, a potent new class of bacterial type II topoisomerase inhibitor by chemical-genetic profiling in Staphylococcus aureus. Chem Biol. 2011;18(8):955-965. doi:10.1016/j.chembiol.2011.06.011 73. Takahashi Y, Nakamura H, Ogata R, Matsuda N, Hamada M, Naganawa H, Takita T, Iitaka Y, Sato K, and Takeuchi T. Kijimicin, a polyether antibiotic. J Antibiot (Tokyo). 1990;43(4):441-443. doi:10.7164/antibiotics.43.441 74. Sergio S, Pirali G, White R, and Parenti F. Lipiarmycin, a new antibiotic from Actinoplanes. III. Mechanism of action. J Antibiot (Tokyo). 1975;28(7):543-

6 References

549. doi:10.7164/antibiotics.28.543 75. Glaus F, and Altmann K-H. Total synthesis of the tiacumicin B (lipiarmycin A3/fidaxomicin) aglycone. Angew Chemie Int Ed. 2015;54(6):1937-1940. doi:10.1002/anie.201409510 76. Kim BM, Choi HY, Kim GW, Zheng CJ, Kim YH, and Kim WG. Madurahydroxylactone, an inhibitor of Staphylococcus aureus FtsZ from Nonomuraea sp. AN100570. J Microbiol Biotechnol. 2017;27(11):1994-1998. doi:10.4014/jmb.1708.08044 77. Marchand C, Beutler JA, Wamiru A, Budihas S, Mollmann U, Heinisch L, Mellors JW, Le Grice SF, and Pommier Y. Madurahydroxylactone derivatives as dual inhibitors of human immunodeficiency virus type 1 integrase and RNase H. Antimicrob Agents Chemother. 2008;52(1):361-364. doi:10.1128/AAC.00883-07 78. Anzai Y, Li S, Chaulagain MR, Kinoshita K, Kato F, Montgomery J, and Sherman DH. Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics. Chem Biol. 2008;15(9):950-959. doi:10.1016/j.chembiol.2008.07.014 79. Liu M, and Douthwaite S. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy. Proc Natl Acad Sci. 2002;99(23):14658-14663. doi:10.1073/pnas.232580599 80. Pokhrel AR, Chaudhary AK, Nguyen HT, Dhakal D, Le TT, Shrestha A, Liou K, and Sohng JK. Overexpression of a pathway specific negative regulator enhances production of daunorubicin in bldA deficient Streptomyces peucetius ATCC 27952. Microbiol Res. 2016;192:96-102. doi:10.1016/j.micres.2016.06.009 81. Sohng JK, Yamaguchi T, Seong CN, Baik KS, Park SC, Lee HJ, Jang SY, Simkhada JR, and Yoo JC. Production, isolation and biological activity of nargenicin from Nocardia sp. CS682. Arch Pharm Res. 2008;31(10):1339- 1345. doi:10.1007/s12272-001-2115-0 82. Coronelli C, Pagani H, Bardone MR, and Lancini GC. Purpuromycin, a new antibiotic isolated from Actinoplanes ianthinogenes n. sp. J Antibiot (Tokyo). 1974;27(3):161-168. doi:10.7164/antibiotics.27.161 83. Kirillov S, Vitali LA, Goldstein BP, Monti F, Semenkov Y, Makhno V, Ripa S, Pon CL, and Gualerzi CO. Purpuromycin: An antibiotic inhibiting tRNA aminoacylation. RNA. 1997;3(8):905-913. 84. Marcone GL, Binda E, Reguzzoni M, Gastaldo L, Dalmastri C, and Marinelli F. Classification of Actinoplanes sp. ATCC 33076, an actinomycete that produces the glycolipodepsipeptide antibiotic ramoplanin, as Actinoplanes ramoplaninifer sp. nov. Int J Syst Evol Microbiol. 2017;67(10):4181-4188. doi:10.1099/ijsem.0.002281 85. McCafferty DG, Cudic P, Frankel BA, Barkallah S, Kruger RG, and Li W. Chemistry and biology of the ramoplanin family of peptide antibiotics. Biopolymers. 2002;66(4):261-284. doi:10.1002/bip.10296 86. Floss HG, and Yu T-W. Rifamycin-Mode of action, resistance, and

6 References

biosynthesis. Chem Rev. 2005;105(2):621-632. doi:10.1021/cr030112j 87. Crowe CC, and Sanders WE. Rosamicin: Evaluation in vitro and comparison with erythromycin and lincomycin. Antimicrob Agents Chemother. 1974;5(3):272-275. doi:10.1128/AAC.5.3.272 88. Siegrist S, Lagouardat J, Moreau N, and Goffic F. Mechanism of action of a 16-membered macrolide. Binding of rosaramicin to the Escherichia coli ribosome and its subunits. Eur J Biochem. 1981;115(2):323-327. doi:10.1111/j.1432-1033.1981.tb05241.x 89. Singh MP, Petersen PJ, Weiss WJ, Kong F, and Greenstein M. Saccharomicins, novel heptadecaglycoside antibiotics produced by Saccharothrix espanaensis: Antibacterial and mechanistic activities. Antimicrob Agents Chemother. 2000;44(8):2154-2159. doi:10.1128/AAC.44.8.2154-2159.2000 90. Kondo J, Koganei M, and Kasahara T. Crystal structure and specific binding mode of sisomicin to the bacterial ribosomal decoding site. ACS Med Chem Lett. 2012;3(9):741-744. doi:10.1021/ml300145y 91. Weinstein M, Wagman G, and Waitz J. Discovery and isolation of sisomicin. Infection. 1976;4(S4):S285-S288. doi:10.1007/BF01646951 92. Somma S, Gastaldo L, and Corti A. Teicoplanin, a new antibiotic from Actinoplanes teichomyceticus nov. sp. Antimicrob Agents Chemother. 1984;26(6):917-923. doi:10.1128/AAC.26.6.917 93. Wang F, Zhou H, Olademehin OP, Kim SJ, and Tao P. Insights into key interactions between vancomycin and bacterial cell wall structures. ACS Omega. 2018;3(1):37-45. doi:10.1021/acsomega.7b01483 94. Donelli G, Vuotto C, and Mastromarino P. Phenotyping and genotyping are both essential to identify and classify a probiotic microorganism. Microb Ecol Heal Dis. 2013;24(0):1-8. doi:10.3402/mehd.v24i0.20105 95. Schumann P, and Pukall R. The discriminatory power of ribotyping as automatable technique for differentiation of bacteria. Syst Appl Microbiol. 2013;36(6):369-375. doi:10.1016/j.syapm.2013.05.003 96. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, Rooney AP, Yi H, Xu X-W, De Meyer S, and Trujillo ME. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol. 2018;68(1):461-466. doi:10.1099/ijsem.0.002516 97. Gaudêncio SP, and Pereira F. Dereplication: Racing to speed up the natural products discovery process. Nat Prod Rep. 2015;32(6):779-810. doi:10.1039/c4np00134f 98. Omura S, Takahashi Y, Iwai Y, and Tanaka H. Kitasatosporia, a new genus of the order Actinomycetales. J Antibiot (Tokyo). 1982;35(8):1013-1019. doi:10.7164/antibiotics.35.1013 99. Vaidya JD, Hornung BVH, Smidt H, Edwards JE, and Plugge CM. Propionibacterium ruminifibrarum sp. nov., isolated from cow rumen fibrous content. Int J Syst Evol Microbiol. 2019;69(8):2584-2590. doi:10.1099/ijsem.0.003544

6 References

100. Simpson PJ, Ross RP, Fitzgerald GF, and Stanton C. Bifidobacterium psychraerophilum sp. nov. and Aeriscardovia aeriphila gen. nov., sp. nov., isolated from a porcine caecum. Int J Syst Evol Microbiol. 2004;54(2):401- 406. doi:10.1099/ijs.0.02667-0 101. Combet-Blanc Y, Thomas P, Ollivier B, Koussémon M, Cayol JL, Garcia JL, and Patel BKC. Propionibacterium microaerophilum sp. nov., a microaerophilic bacterium isolated from olive mill wastewater. Int J Syst Evol Microbiol. 2001;51(4):1373-1382. doi:10.1099/00207713-51-4-1373 102. Shirling EB, and Gottlieb D. Methods for characterization of Streptomyces species. Int J Syst Evol Microbiol. 1966;16(3):313-340. 103. Wink J, Schumann P, Atasayar E, Klenk HP, Zaburannyi N, Westermann M, Martin K, Glaeser SP, and Kämpfer P. ‘Streptomyces caelicus’, an antibiotic- producing species of the genus Streptomyces, and Streptomyces canchipurensis Li et al. 2015 are later heterotypic synonyms of Streptomyces muensis Ningthoujam et al. 2014. Int J Syst Evol Microbiol. 2017;67(3):548- 556. doi:10.1099/ijsem.0.001612 104. Pridham TG, and Gottlieb D. The utilization of carbon compounds by some actinomycetales as an aid for species determination. J Bacteriol. 1948;56(1):107-114. 105. Kämpfer P, Huber B, Buczolits S, Thummes K, Grün-Wollny I, and Busse HJ. Streptomyces specialis sp. nov. Int J Syst Evol Microbiol. 2008;58(11):2602-2606. doi:10.1099/ijs.0.2008/001008-0 106. Chaiya L, Matsumoto A, Wink J, Inahashi Y, Risdian C, Pathom-aree W, and Lumyong S. Amycolatopsis eburnea sp. nov., an actinomycete associated with arbuscular mycorrhizal fungal spores. Int J Syst Evol Microbiol. August 2019:1-6. doi:10.1099/ijsem.0.003669 107. Goodfellow M, Busarakam K, Idris H, Labeda DP, Nouioui I, Brown R, Kim BY, del Carmen Montero-Calasanz M, Andrews BA, and Bull AT. Streptomyces asenjonii sp. nov., isolated from hyper-arid Atacama Desert soils and emended description of Streptomyces viridosporus Pridham et al. 1958. Antonie van Leeuwenhoek, Int J Gen Mol Microbiol. 2017;110(9):1133- 1148. doi:10.1007/s10482-017-0886-7 108. Lechevalier MP, and Lechevalier H. Chemical composition as a criterion in the classification of aerobic actinomycetes. Int J Syst Bacteriol. 1970;20(4):435-443. 109. Landwehr W, Kämpfer P, Glaeser SP, Rückert C, Kalinowski J, Blom J, Goesmann A, Mack M, Schumann P, Atasayar E, Hahnke RL, Rohde M, Martin K, Stadler M, and Wink J. Taxonomic analyses of members of the Streptomyces cinnabarinus cluster, description of Streptomyces cinnabarigriseus sp. nov. and Streptomyces davaonensis sp. nov. Int J Syst Evol Microbiol. 2018;68(1):382-393. doi:10.1099/ijsem.0.002519 110. Pan HQ, Zhang DF, Li L, Jiang Z, Cheng J, Zhang YG, Wang HF, Hu JC, and Li WJ. Nocardiopsis oceani sp. nov. and Nocardiopsis nanhaiensis sp. nov., actinomycetes isolated from marine sediment of the South China Sea. Int J Syst Evol Microbiol. 2015;65(10):3384-3391. doi:10.1099/ijsem.0.000425

6 References

111. Collins MD, and Shah HN. Fatty acid, menaquinone and polar lipid composition of Rothia dentocariosa. Arch Microbiol. 1984;137(3):247-249. 112. Nurkanto A, Lisdiyanti P, Hamada M, Ratnakomala S, Shibata C, and Tamura T. Actinoplanes bogoriensis sp. nov., a novel actinomycete isolated from leaf litter. J Antibiot (Tokyo). 2016;69(1):26-30. doi:10.1038/ja.2015.81 113. Lechevalier MP, De Bievre C, and Lechevalier H. Chemotaxonomy of aerobic Actinomycetes: Phospholipid composition. Biochem Syst Ecol. 1977;5(4):249-260. doi:10.1016/0305-1978(77)90021-7 114. Collins MD, Pirouz T, Goodfellow M, and Minnikin DE. Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol. 1977;100(2):221-230. doi:10.1099/00221287-100-2-221 115. Loucif L, Bendjama E, Gacemi-Kirane D, and Rolain J-M. Rapid identification of Streptomyces isolates by MALDI-TOF MS. Microbiol Res. 2014;169(12):940-947. doi:10.1016/j.micres.2014.04.004 116. Wink J, Schumann P, Spoer C, Eisenbarth K, Glaeser SP, Martin K, and Kampfer P. Emended description of Actinoplanes friuliensis and description of Actinoplanes nipponensis sp. nov., antibiotic-producing species of the genus Actinoplanes. Int J Syst Evol Microbiol. 2014;64(Pt 2):599-606. doi:10.1099/ijs.0.057836-0 117. Schumann P, and Maier T. MALDI-TOF Mass Spectrometry Applied to Classification and Identification of Bacteria. Vol 41. (Goodfellow M, Sutcliffe I, and Chun J, eds.). Academic Press; 2014. doi:10.1016/bs.mim.2014.06.002 118. Mesbah M, Premachandran U, and Whitman WB. Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol. 1989;39(2):159-167. doi:10.1099/00207713-39-2-159 119. Meier-Kolthoff JP, Klenk HP, and Göker M. Taxonomic use of DNA G+C content and DNA-DNA hybridization in the genomic age. Int J Syst Evol Microbiol. 2014;64(PART 2):352-356. doi:10.1099/ijs.0.056994-0 120. Gao B, and Gupta RS. Phylogenetic framework and molecular signatures for the main clades of the phylum Actinobacteria. Microbiol Mol Biol Rev. 2012;76(1):66-112. doi:10.1128/MMBR.05011-11 121. Fox G, Stackebrandt E, Hespell R, et al. The phylogeny of prokaryotes. Science (80- ). 1980;209(4455):457-463. doi:10.1126/science.6771870 122. Monciardini P, Sosio M, Cavaletti L, Chiocchini C, and Donadio S. New PCR primers for the selective amplification of 16S rDNA from different groups of actinomycetes. FEMS Microbiol Ecol. 2002;42(3):419-429. doi:10.1111/j.1574-6941.2002.tb01031.x 123. Woese CR, and Fox GE. Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc Natl Acad Sci. 1977;74(11):5088-5090. doi:10.1073/pnas.74.11.5088 124. Tang B, Yu Y, Zhi X, Yang L, Cen X, Zhao G, and Ding X. Streptomyces yangpuensis sp. nov., isolated from soil. Int J Syst Evol Microbiol.

6 References

2016;66(3):1224-1229. doi:10.1099/ijsem.0.000861 125. le Roes-Hill M, and Meyers PR. Streptomyces polyantibioticus sp. nov., isolated from the banks of a river. Int J Syst Evol Microbiol. 2009;59(6):1302- 1309. doi:10.1099/ijs.0.006171-0 126. Kaewkla O, and Franco CMM. Streptomyces roietensis sp. nov., an endophytic actinobacterium isolated from the surface-sterilized stem of jasmine rice, Oryza sativa KDML 105. Int J Syst Evol Microbiol. 2017;67(11):4868-4872. doi:10.1099/ijsem.0.002402 127. Mohammadipanah F, Hamedi J, Spröer C, Rohde M, Montero-Calasanz M del C, and Klenk HP. Streptomyces zagrosensis sp. nov., isolated from soil. Int J Syst Evol Microbiol. 2014;64(2014):3434-3440. doi:10.1099/ijs.0.064527-0 128. Brosius J, Palmer ML, Kennedy PJ, and Noller HF. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci. 1978;75(10):4801-4805. doi:10.1073/pnas.75.10.4801 129. Ritacco F V, Haltli B, Janso JE, Greenstein M, and Bernan VS. Dereplication of Streptomyces soil isolates and detection of specific biosynthetic genes using an automated ribotyping instrument. J Ind Microbiol Biotechnol. 2003;30(8):472-479. doi:10.1007/s10295-003-0038-0 130. Rong X, and Huang Y. Taxonomic evaluation of the Streptomyces hygroscopicus clade using multilocus sequence analysis and DNA-DNA hybridization, validating the MLSA scheme for systematics of the whole genus. Syst Appl Microbiol. 2012;35:7-18. doi:10.1016/j.syapm.2011.10.004 131. Rong X, Guo Y, and Huang Y. Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA- DNA hybridization, and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens. Syst Appl Microbiol. 2009;32(5):314-322. doi:10.1016/j.syapm.2009.05.003 132. Chun J, and Rainey FA. Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea. Int J Syst Evol Microbiol. 2014;64(PART 2):316-324. doi:10.1099/ijs.0.054171-0 133. Tsai M-H, Liu Y-Y, Soo V-W, and Chen C-C. A new genome-to-genome comparison approach for large-scale revisiting of current microbial taxonomy. Microorganisms. 2019;7(6):161. doi:10.3390/microorganisms7060161 134. Beutler JA, Alvarado AB, Schaufelberger DE, Andrews P, and McCloud TG. Dereplication of phorbol bioactives: Lyngbya majuscula and Croton cuneatus. J Nat Prod. 1990;53(4):867-874. doi:10.1021/np50070a014 135. Hubert J, Nuzillard JM, and Renault JH. Dereplication strategies in natural product research: How many tools and methodologies behind the same concept? Phytochem Rev. 2017;16(1):55-95. doi:10.1007/s11101-015-9448-7 136. Bucar F, Wube A, and Schmid M. Natural product isolation – how to get from biological material to pure compounds. Nat Prod Rep. 2013;30(4):525. doi:10.1039/c3np20106f 137. Bauer A, Kirby W, Sherris J, and Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 1966;45(4):493-496.

6 References

138. Charousová I, Javoreková S, and Wink J. Isolation and characterization of Streptomyces rishiriensis (vy31) with antibiotic activity against various pathogenic microorganisms. J Microbiol Biotechnol Food Sci. 2015;04(Special 01):23-27. doi:10.15414/jmbfs.2015.4.special1.23-27 139. Bouslimani A, Sanchez LM, Garg N, and Dorrestein PC. Mass spectrometry of natural products: Current, emerging and future technologies. Nat Prod Rep. 2014;31(6):718. doi:10.1039/c4np00044g 140. Breton RC, and Reynolds WF. Using NMR to identify and characterize natural products. Nat Prod Rep. 2013;30(4):501. doi:10.1039/c2np20104f 141. NIAID. Hepatitis. https://www.niaid.nih.gov/diseases-conditions/hepatitis. Published 2019. Accessed October 14, 2019. 142. Nainan O V, Xia G, Vaughan G, and Margolis HS. Diagnosis of hepatitis A virus infection: A molecular approach. Clin Microbiol Rev. 2006;19(1):63-79. doi:10.1128/CMR.19.1.63-79.2006 143. Schaefer S. Hepatitis B virus taxonomy and hepatitis B virus genotypes. World J Gastroenterol. 2007;13(1):14. doi:10.3748/wjg.v13.i1.14 144. Pietschmann T, Kaul A, Koutsoudakis G, Shavinskaya A, Kallis S, Steinmann E, Abid K, Negro F, Dreux M, Cosset F-L, and Bartenschlager R. Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci. 2006;103(19):7408-7413. doi:10.1073/pnas.0504877103 145. Botelho-Souza LF, Vasconcelos MPA, dos Santos A de O, Salcedo JMV, and Vieira DS. Hepatitis delta: virological and clinical aspects. Virol J. 2017;14(1):177. doi:10.1186/s12985-017-0845-y 146. Doceul V, Bagdassarian E, Demange A, and Pavio N. Zoonotic Hepatitis E Virus: Classiﬁcation, Animal Reservoirs and Transmission Routes. Viruses. 2016;8(10):270. doi:10.3390/v8100270 147. Landwehr W. Isolation and characterization of novel Actinobacteria and myxobacteria especially from marine habitats. 2017. 148. Heuer H, Krsek M, Baker P, and Smalla K. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel- electrophoretic separation in denaturing gradients. Appl Environ Microbiol. 1997;63(8):3233-3241. 149. Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E, and Goodfellow M, eds. Nucleic Acid Techniques in Bacterial Systematics. Chichester, UK: Wiley; 1991:115–176. 150. Mohr KI, Moradi A, Glaeser SP, Kämpfer P, Gemperlein K, Nübel U, Schumann P, Müller R, and Wink J. Nannocystis konarekensis sp. nov., a novel myxobacterium from an Iranian desert. Int J Syst Evol Microbiol. 2018;68(3):721-729. doi:10.1099/ijsem.0.002569 151. Guo YP, Zheng W, Rong XY, and Huang Y. A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: Use of multilocus sequence analysis for streptomycete systematics. Int J Syst Evol Microbiol. 2008;58(1):149-159. doi:10.1099/ijs.0.65224-0

100

6 References

152. Labeda DP, Dunlap CA, Rong X, Huang Y, Doroghazi JR, Ju K-S, and Metcalf WW. Phylogenetic relationships in the family Streptomycetaceae using multi-locus sequence analysis. Antonie Van Leeuwenhoek. 2017;110(4):563-583. doi:10.1007/s10482-016-0824-0 153. Hayakawa M, Otoguro M, Takeuchi T, Yamazaki T, and Iimura Y. Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter. Antonie van Leeuwenhoek, Int J Gen Mol Microbiol. 2000;78(2):171-185. doi:10.1023/A:1026579426265 154. Landwehr W, Karwehl S, Schupp PJ, Schumann P, and Wink J. Biological active rakicidins A , B and E produced by the marine Micromonospora sp . isolate Guam1582. Adv Biotech Micro. 2016;1(2):555558. doi:10.19080/AIBM.2016.01.555558 155. Mohr KI, Stechling M, Wink J, Wilharm E, and Stadler M. Comparison of myxobacterial diversity and evaluation of isolation success in two niches: Kiritimati Island and German compost. Microbiologyopen. 2016;5(2):268- 278. doi:10.1002/mbo3.325 156. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, and Chun J. Introducing EzBioCloud: A taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol. 2017;67(5):1613- 1617. doi:10.1099/ijsem.0.001755 157. Thompson JD, Higgins DG, and Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22(22):4673-4680. doi:10.1093/nar/22.22.4673 158. Kumar S, Stecher G, Li M, Knyaz C, and Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547-1549. doi:10.1093/molbev/msy096 159. Felsenstein J. Evolutionary trees from DNA sequences: A maximum likelihood approach. 1981;17:368-376. doi:10.1007/BF01734359 160. Fitch WM. Toward defining the course of evolution: Minimum change for a specific tree topology. Syst Zool. 1971;20(4):406-416. doi:10.2307/2412116 161. Saitou N, and Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406-425. 162. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution (N Y). 1985;39(4):783-791. doi:10.2307/2408678 163. Suter M. Isolierung und charakterisierung von melanin-negativen mutanten aus Streptomyces glaucescens: Thesis no. 6276. 1978. 164. Wink J. Polyphasic taxonomy and antibiotic formation in some closely related genera of the family Pseudonocardiaceae. In: Pandelai SG, ed. Recent Research Development in Antibiotics. Kerala, India: Transworld Research Network; 2003:97-140. 165. Kutzner H. The family Streptomycetaceae. In: Starr M, Stolp H, Trüper H, Balons A, and Schlegel H, eds. The Prokaryotes – A Handbook on Habitats,

101

6 References

Isolation and Identification of Bacteria. Springer Verlag, Berlin; 1981:2028- 2090. 166. Humble MW, King A, and Phillips I. API ZYM : a simple rapid system for the detection of bacterial enzymes. J Clin Pathol. 1977;30(3):275-277. 167. Soto A, Zapardiel J, and Soriano F. Evaluation of API Coryne system for identifying coryneform bacteria. J Clin Pathol. 1994;47:756-759. 168. Staneck JL, and Roberts GD. Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl Microbiol. 1974;28(2):226-231. 169. Minnikin DE, O’Donnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, and Parlett JH. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods. 1984;2(5):233- 241. doi:10.1016/0167-7012(84)90018-6 170. Minnikin DE, Patel P V., Alshamaony L, and Goodfellow M. Polar lipid composition in the classification of Nocardia and related bacteria. Int J Syst Bacteriol. 1977;27(2):104-117. 171. Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Tech Note. 1990;101:MIDI Inc., Newark, DE. 172. Rong X, and Huang Y. Taxonomic evaluation of the Streptomyces griseus clade using multilocus sequence analysis and DNA - DNA hybridization, with proposal to combine 29 species and three subspecies as 11 genomic species. Int J Syst Evol Microbiol. 2010;60(3):696-703. doi:10.1099/ijs.0.012419-0 173. Tamaoka J, and Komagata K. Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS MicrobiolLetters. 1984;25:125-128. 174. Edgar RC. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792-1797. doi:10.1093/nar/gkh340 175. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980;16(2):111-120. doi:10.1007/BF01731581 176. Rupcic Z, Chepkirui C, Hernández-Restrepo M, Crous PW, Luangsa-ard JJ, and Stadler M. New nematicidal and antimicrobial secondary metabolites from a new species in the new genus, Pseudobambusicola thailandica. MycoKeys. 2018;33:1-23. doi:10.3897/mycokeys.33.23341 177. Mulwa LS, Jansen R, Praditya DF, Mohr KI, Okanya PW, Wink J, Steinmann E, and Stadler M. Lanyamycin, a macrolide antibiotic from Sorangium cellulosum, strain Soce 481 (Myxobacteria). Beilstein J Org Chem. 2018;14:1554-1562. doi:10.3762/bjoc.14.132 178. Tamura K, and Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993;10(3):512-526. doi:10.1093/oxfordjournals.molbev.a040023 179. Altschul SF, Gish W, Miller W, Myers EW, and Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403-410. doi:10.1016/S0022-

102

6 References

2836(05)80360-2 180. Cebrián G, Condón S, and Mañas P. Physiology of the inactivation of vegetative bacteria by thermal treatments: Mode of action, influence of environmental factors and inactivation kinetics. Foods. 2017;6(12):107. doi:10.3390/foods6120107 181. Bobek J, Šmídová K, and Čihák M. A waking review: Old and novel insights into the spore germination in Streptomyces. Front Microbiol. 2017;8(NOV):1- 12. doi:10.3389/fmicb.2017.02205 182. Küster E, and Williams ST. Selection of media for isolation of Streptomycetes. Nature. 1964;202(4935):928-929. doi:10.1038/202928a0 183. Webb M. The influence of magnesium on cell division: The effect of magnesium on the growth and cell division of various bacterial species in complex media. J Gen Microbiol. 1949;3(3):410-417. doi:10.1099/00221287- 3-3-410 184. Pedrini AM, Geroldi D, Siccardi A, and Falaschi A. Studies on the mode of action of nalidixic acid. Eur J Biochem. 1972;25(2):359-365. doi:10.1111/j.1432-1033.1972.tb01704.x 185. Cozzarelli NR. The mechanism of action of inhibitors of DNA synthesis. Annu Rev Biochem. 1977;46(1):641-668. doi:10.1146/annurev.bi.46.070177.003233 186. Salkin IF, and Hurd N. Quantitative evaluation of the antifungal properties of cycloheximide. Antimicrob Agents Chemother. 1972;1(3):177-184. doi:10.1128/AAC.1.3.177 187. Schneider-Poetsch T, Ju J, Eyler DE, Dang Y, Bhat S, Merrick WC, Green R, Shen B, and Liu JO. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol. 2010;6(3):209-217. doi:10.1038/nchembio.304 188. Hayakawa M, Sadakata T, Kajiura T, and Nonomura H. New methods for the highly selective isolation of Micromonospora and Microbispora from soil. J Ferment Bioeng. 1991;72(5):320-326. doi:10.1016/0922-338X(91)90080-Z 189. Hayakawa M, and Nonomura H. Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. J Ferment Technol. 1987;65(5):501-509. doi:10.1016/0385-6380(87)90108-7 190. Ichikawa N, Oguchi A, Ikeda H, et al. Genome sequence of Kitasatospora setae NBRC 14216T: An evolutionary snapshot of the family Streptomycetaceae. DNA Res. 2010;17(6):393-406. doi:10.1093/dnares/dsq026 191. Busarakam K, Bull AT, Girard G, Labeda DP, Van Wezel GP, and Goodfellow M. Streptomyces leeuwenhoekii sp. nov., the producer of chaxalactins and chaxamycins, forms a distinct branch in Streptomyces gene trees. Antonie van Leeuwenhoek, Int J Gen Mol Microbiol. 2014;105(5):849- 861. doi:10.1007/s10482-014-0139-y 192. Kämpfer P, Huber B, Buczolits S, Thummes K, Grün-Wollny I, and Busse HJ. Streptomyces specialis sp. nov. Int J Syst Evol Microbiol.

103

6 References

2008;58(11):2602-2606. doi:10.1099/ijs.0.2008/001008-0 193. Ayed A, Slama N, Mankai H, Bachkouel S, ElKahoui S, Tabbene O, and Limam F. Streptomyces tunisialbus sp. nov., a novel Streptomyces species with antimicrobial activity. Antonie Van Leeuwenhoek. 2018;111(9):1571- 1581. doi:10.1007/s10482-018-1046-4 194. Schnermann MJ, and Boger DL. Total synthesis of piericidin A1 and B1. J Am Chem Soc. 2005;127(45):15704-15705. doi:10.1021/ja055041f 195. Jeng M, Hall C, Crane FL, Takahashi N, Tamura S, and Folkers K. Inhibition of mitochondrial electron transport by piericidin A and related compounds. Biochemistry. 1968;7(4):1311-1322. doi:10.1021/bi00844a010 196. Ooka K, Fukumoto A, Yamanaka T, and Shimada K. Piericidins, novel quorum-sensing inhibitors against Chromobacterium violaceum CV026, from Streptomyces sp. TOHO-Y209 and TOHO-O348. Open J Med Chem. 2013;3(December):93-99. 197. Heikkilä J, and Akerman K. (-)-Indolactam V activates protein kinase C and induces changes in muscarinic receptor functions in SH-SY5Y human neuroblastoma cells. Biochem Biophys Res Commun. 1989;162(3). doi:10.1016/0006-291X(89)90802-4 198. Chen S, Borowiak M, Fox JL, Maehr R, Osafune K, Davidow L, Lam K, Peng LF, Schreiber SL, Rubin LL, and Melton D. A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat Chem Biol. 2009;5(4):258-265. doi:10.1038/nchembio.154 199. Irie K, Hirota M, Hagiwara N, Koshimizu K, Hayashi H, Murao S, Tokuda H, and Ito Y. The Epstein-Barr virus early antigen inducing indole alkaloids, (— )-indolactam V and its related compounds, produced by actinomycetes. Agric Biol Chem. 1984;48(5):1269-1274. doi:10.1080/00021369.1984.10866289 200. Irie K, Tomimatsu S, Nakagawa Y, Ohigashi H, and Hayashi H. Isolation of (- )-14-O-malonylindolactam-V as a possible precursor of (-)-indolactam-V and (-)-14-O-acetylindolactam-V from Streptomyces blastmyceticum. Biosci Biotechnol Biochem. 1999;63(9):1669-1670. doi:10.1271/bbb.63.1669 201. Macherla VR, Liu J, Bellows C, Teisan S, Nicholson B, Lam KS, and Potts BCM. Glaciapyrroles A, B, and C, pyrrolosesquiterpenes from a Streptomyces sp. isolated from an Alaskan marine sediment. J Nat Prod. 2005;68(5):780- 783. doi:10.1021/np049597c 202. Strom K, Sjogren J, Broberg A, and Schnurer J. Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid. Appl Environ Microbiol. 2002;68(9):4322-4327. doi:10.1128/AEM.68.9.4322-4327.2002 203. Jiang W, Zhou W, Uchida H, Kikumori M, Irie K, Watanabe R, Suzuki T, Sakamoto B, Kamio M, and Nagai H. A New Lyngbyatoxin from the Hawaiian cyanobacterium Moorea producens. Mar Drugs. 2014;12(5):2748- 2759. doi:10.3390/md12052748 204. Takahashi Y. Genus Kitasatospora, taxonomic features and diversity of secondary metabolites. J Antibiot (Tokyo). 2017;70(5):506-513. doi:10.1038/ja.2017.8

104

6 References

205. da Silva LJ, Taketani RG, de Melo IS, Goodfellow M, and Zucchi TD. Streptomyces araujoniae sp. nov.: an actinomycete isolated from a potato tubercle. Antonie Van Leeuwenhoek. 2013;103(6):1235-1244. doi:10.1007/s10482-013-9901-9 206. Vargas Hoyos HA, Nobre Santos S, Da Silva LJ, Paulino Silva FS, Bonaldo Genuário D, Domingues Zucchi T, and Melo IS. Streptomyces rhizosphaericola sp. nov., an actinobacterium isolated from the wheat rhizosphere. Int J Syst Evol Microbiol. 2019;69(8):2431-2439. doi:10.1099/ijsem.0.003498 207. Dröse S, and Altendorf K. Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases. J Exp Biol. 1997;200(1):1-8. 208. Kretschmer A, Dorgerloh M, Deeg M, and Hagenmaier H. The structures of novel insecticidal macrolides: Bafilomycins D and E, and oxohygrolidin. Agric Biol Chem. 1985;49(8):2509-2511. doi:10.1271/bbb1961.49.2509 209. Yu Z, Zhao L-X, Jiang C-L, Duan Y, Wong L, Carver KC, Schuler LA, and Shen B. Bafilomycins produced by an endophytic actinomycete Streptomyces sp. YIM56209. J Antibiot (Tokyo). 2011;64(1):159-162. doi:10.1038/ja.2010.147 210. Ding N, Jiang Y, Han L, Chen X, Ma J, Qu X, Mu Y, Liu J, Li L, Jiang C, and Huang X. Bafilomycins and odoriferous sesquiterpenoids from Streptomyces albolongus isolated from Elephas maximus feces. J Nat Prod. 2016;79(4):799-805. doi:10.1021/acs.jnatprod.5b00827 211. Yeganeh B, Ghavami S, Kroeker AL, Mahood TH, Stelmack GL, Klonisch T, Coombs KM, and Halayko AJ. Suppression of influenza A virus replication in human lung epithelial cells by noncytotoxic concentrations bafilomycin A1. Am J Physiol Cell Mol Physiol. 2015;308(3):L270-L286. doi:10.1152/ajplung.00011.2014 212. Tang B, Xie F, Zhao W, Wang J, Dai S, Zheng H, Ding X, Cen X, Liu H, Yu Y, Zhou H, Zhou Y, Zhang L, Goodfellow M, and Zhao G-P. A systematic study of the whole genome sequence of Amycolatopsis methanolica strain 239 T provides an insight into its physiological and taxonomic properties which correlate with its position in the genus. Synth Syst Biotechnol. 2016;1(3):169- 186. doi:10.1016/j.synbio.2016.05.001 213. Labeda DP, Goodfellow M, Chun J, Zhi XY, and Li WJ. Reassessment of the systematics of the suborder Pseudonocardineae: Transfer of the genera within the family Actinosynnemataceae Labeda and Kroppenstedt 2000 emend. Zhi et al. 2009 into an emended family Pseudonocardiaceae Emble. Int J Syst Evol Microbiol. 2011;61(6):1259-1264. doi:10.1099/ijs.0.024984-0 214. Kim SB, and Goodfellow M. Reclassification of Amycolatopsis rugosa Lechevalier et al. 1986 as Prauserella rugosa gen. nov., comb. nov. Int J Syst Bacteriol. 1999;49(2):507-512. doi:10.1099/00207713-49-2-507 215. Tan GYA, Ward AC, and Goodfellow M. Exploration of Amycolatopsis diversity in soil using genus-specific primers and novel selective media. Syst Appl Microbiol. 2006;29(7):557-569. doi:10.1016/j.syapm.2006.01.007 216. Xu L, Huang H, Wei W, Zhong Y, Tang B, Yuan H, Zhu L, Huang W, Ge M,

105

6 References

Yang S, Zheng H, Jiang W, Chen D, Zhao G-P, and Zhao W. Complete genome sequence and comparative genomic analyses of the vancomycin- producing Amycolatopsis orientalis. BMC Genomics. 2014;15(1):363. doi:10.1186/1471-2164-15-363 217. Nadkarni SR, Patel M V., Chatterjee S, Vijayakumar EKS, Desikan KR, Blumbach J, and Ganguli BN. Balhimycin, a new glycopeptide antibiotic produced by Amycolatopsis sp. Y-86,21022. Taxonomy, production, isolation and biological activity. J Antibiot (Tokyo). 1994;47(3):334-341. doi:10.7164/antibiotics.47.334 218. August PR, Tang L, Yoon YJ, Ning S, Müller R, Yu T-W, Taylor M, Hoffmann D, Kim C-G, Zhang X, Hutchinson CR, and Floss HG. Biosynthesis of the ansamycin antibiotic rifamycin: Deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem Biol. 1998;5(2):69-79. doi:10.1016/S1074- 5521(98)90141-7 219. Wink JM, Kroppenstedt RM, Ganguli BN, Nadkarni SR, Schumann P, Seibert G, and Stackebrandt E. Three new antibiotic producing species of the genus Amycolatopsis, Amycolatopsis balhimycina sp. nov., A. tolypomycina sp. nov., A. vancoresmycina sp. nov. Syst Appl Microbiol. 2003;26(1):38-46. doi:10.1078/072320203322337290 220. Mehta D, and Satyanarayana T. Bacterial and archaeal α-amylases: Diversity and amelioration of the desirable characteristics for industrial applications. Front Microbiol. 2016;7(JUL):1-21. doi:10.3389/fmicb.2016.01129 221. Zucchi TD, Tan GYA, Bonda ANV, Frank S, Kshetrimayum JD, and Goodfellow M. Amycolatopsis granulosa sp. nov., Amycolatopsis ruanii sp. nov. and Amycolatopsis thermalba sp. nov., thermophilic actinomycetes isolated from arid soils. Int J Syst Evol Microbiol. 2012;62(Pt 6):1245-1251. doi:10.1099/ijs.0.031039-0 222. Dailey HA, Gerdes S, Dailey TA, Burch JS, and Phillips JD. Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin. Proc Natl Acad Sci. 2015;112(7):2210-2215. doi:10.1073/pnas.1416285112 223. Zhu L, Qian X, Chen D, and Ge M. Role of two 5-aminolevulinic acid biosynthetic pathways in heme and secondary metabolite biosynthesis in Amycolatopsis orientalis. J Basic Microbiol. 2018;58(2):198-205. doi:10.1002/jobm.201600758 224. Cleary JL, Kolachina S, Wolfe BE, and Sanchez LM. Coproporphyrin III produced by the bacterium Glutamicibacter arilaitensis binds zinc and is ipregulated by fungi in cheese rinds. Tullman-Ercek D, ed. mSystems. 2018;3(4):1-15. doi:10.1128/mSystems.00036-18 225. Toriya M, Yaginuma S, Murofushi S, Ogawa K, Muto N, Hayashi M, and Matsumoto K. Zincphyrin, a novel coproporphyrin III with zinc from Streptomyces sp. J Antibiot (Tokyo). 1993;46(1):196-200. doi:10.7164/antibiotics.46.196 226. Nishio M, Hasegawa M, Suzuki K, Sawada Y, Hook DJ, and Oki T. Nitrosoxacins A, B and C, new 5-lipoxygenase inhibitors. J Antibiot (Tokyo).

106

6 References

1993;46(1):193-195. doi:10.7164/antibiotics.46.193 227. Rask-Madsen J, Bukhave K, Laursen LS, and Lauritsen K. 5-Lipoxygenase inhibitors for the treatment of inflammatory bowel disease. Agents Actions. 1992;36(S1):C37-C46. doi:10.1007/BF01991022

107

7 Appendix

7 Appendix Table S 1. List of Actinobacteria strains isolated from Indonesian samples

No. Strain Samples location No. Strain Samples location 1 4421 Beach, Bali 99 196831CR Botanical Garden, Bali 2 4422 Beach, Bali 100 196833CR Botanical Garden, Bali 3 4423 Beach, Bali 101 196921CR Botanical Garden, Bali 4 4431 Beach, Bali 102 196932CR Botanical Garden, Bali 5 4433 Beach, Bali 103 197011CR Botanical Garden, Bali 6 4435 Beach, Bali 104 197012CR Botanical Garden, Bali 7 5931 Lava beach, Bali 105 197013CR Botanical Garden, Bali 8 19821 Botanical Garden, 106 197014CR Botanical Garden, Bali Bogor, West Java 9 19823 Botanical Garden, 107 197017CR Botanical Garden, Bali Bogor, West Java 10 19824 Botanical Garden, 108 197019CR Botanical Garden, Bali Bogor, West Java 11 190221 Forest low altitude, 109 197212CR Botanical Garden, Bali Kendari, Southeast Sulawesi 12 190224 Forest low altitude, 110 1982-1 Botanical Garden, Kendari, Southeast Bogor, West Java Sulawesi 13 190225 Forest low altitude, 111 1982-3 Botanical Garden, Kendari, Southeast Bogor, West Java Sulawesi 14 190231 Forest low altitude, 112 1982-3 Botanical Garden, Kendari, Southeast (orange) Bogor, West Java Sulawesi 15 190232 Forest low altitude, 113 1982-4 Botanical Garden, Kendari, Southeast Bogor, West Java Sulawesi 16 190233 Forest low altitude, 114 198311CR Botanical Garden, Kendari, Southeast Bogor, West Java Sulawesi 17 190234 Forest low altitude, 115 198331CR Botanical Garden, Kendari, Southeast Bogor, West Java Sulawesi 18 190235 Forest low altitude, 116 198332CR Botanical Garden, Kendari, Southeast Bogor, West Java Sulawesi 19 190401 Mangrove, Kendari, 117 198333CR Botanical Garden, Southeast Sulawesi Bogor, West Java 20 194601 Cultural Park, Bali 118 198334CR Botanical Garden, Bogor, West Java 21 195105 Cultural Park, Bali 119 198335CR Botanical Garden, Bogor, West Java 22 195107 Cultural Park, Bali 120 198414CR Botanical Garden, Bogor, West Java 23 1951016 Cultural Park, Bali 121 1-SO1 Mangrove, Jakarta 24 180811CR Malang, East Java 122 2112-2 Mangrove, Jakarta 25 180812CR Malang, East Java 123 2112-SO Mangrove, Jakarta

108

7 Appendix

26 180813CR Malang, East Java 124 2113SO Mangrove, Jakarta 27 180814CR Malang, East Java 125 2114-1SO Mangrove, Jakarta 28 1808210CR Malang, East Java 126 2115SO Mangrove, Jakarta 29 180821CR Malang, East Java 127 2118-1 Mangrove, Jakarta 30 180822CR Malang, East Java 128 2118-3 Mangrove, Jakarta 31 180823CR Malang, East Java 129 2118SO Mangrove, Jakarta 32 180824CR Malang, East Java 130 2120SO Mangrove, Jakarta 33 180825CR Malang, East Java 131 2122-2 Mangrove, Jakarta 34 180826CR Malang, East Java 132 2122-5 Mangrove, Jakarta 35 180827CR Malang, East Java 133 2123-1BSO Mangrove, Jakarta 36 180828CR Malang, East Java 134 2124-1SO Mangrove, Jakarta 37 180829CR Malang, East Java 135 2125-2 (grey) Mangrove, Jakarta 38 189921CR Forest low altitude, 136 2125-2 (white) Mangrove, Jakarta Kendari, Southeast Sulawesi 39 189923ACR Forest low altitude, 137 2125-3 Mangrove, Jakarta Kendari, Southeast Sulawesi 40 189923BCR Forest low altitude, 138 2125-5 Mangrove, Jakarta Kendari, Southeast Sulawesi 41 190122BCR Forest low altitude, 139 2126-1SO Mangrove, Jakarta Kendari, Southeast Sulawesi 42 190131ACR Forest low altitude, 140 2126-2SO Mangrove, Jakarta Kendari, Southeast Sulawesi 43 190222CR Forest low altitude, 141 2126-3SO Mangrove, Jakarta Kendari, Southeast Sulawesi 44 190224CR Forest low altitude, 142 2126SO Mangrove, Jakarta Kendari, Southeast Sulawesi 45 190227CR Forest low altitude, 143 2126SO(3) Mangrove, Jakarta Kendari, Southeast Sulawesi 46 190711GyCR Cimahi, West Java 144 2126SO-1 Mangrove, Jakarta 47 190712WCR Cimahi, West Java 145 2126SO-2 Mangrove, Jakarta 48 190721CR Cimahi, West Java 146 2127SO Mangrove, Jakarta 49 194813CR Cultural Park, Bali 147 2-1MKBSO Mangrove, West Kalimantan 50 194821CR Cultural Park, Bali 148 2-2MKBSO Mangrove, West Kalimantan 51 194822CR Cultural Park, Bali 149 3-BBSO Beach, Bali 52 194823CR Cultural Park, Bali 150 4-1BKBSO Beach, West Kalimantan 53 194831CR Cultural Park, Bali 151 4-2BKBSO Beach, West Kalimantan 54 194912CR Cultural Park, Bali 152 4-3BKBSO Beach, West Kalimantan 55 194921CR Cultural Park, Bali 153 4-4BKBSO Beach, West Kalimantan 56 194931CR Cultural Park, Bali 154 4-5BKBSO Beach, West Kalimantan 57 194933RCR Cultural Park, Bali 155 4-6BKBSO Beach, West Kalimantan 58 194934CR Cultural Park, Bali 156 5-1BLSO Beach, Lampung

109

7 Appendix

59 194935CR Cultural Park, Bali 157 5-2BLSO Beach, Lampung 60 194938CR Cultural Park, Bali 158 5-3BLSO Beach, Lampung 61 195003R Cultural Park, Bali 159 6-2BPSO Beach, Papua 62 195211CR Cultural Park, Bali 160 6BPSO Beach, Papua 63 195213BCR Cultural Park, Bali 161 7BPSO Beach, Papua 64 195213CR Cultural Park, Bali 162 9BLSSO Beach, Lampung 65 195215CR Cultural Park, Bali 163 BLH 12-3 Bitung, North Sulawesi 66 195217CR Cultural Park, Bali 164 C190221 Forest low altitude, Kendari, Southeast Sulawesi 67 195218CR Cultural Park, Bali 165 C194911 Cultural Park, Bali 68 195219CR Cultural Park, Bali 166 C194922A Cultural Park, Bali 69 1952211CR Cultural Park, Bali 167 C1950A Cultural Park, Bali 70 1952212CR Cultural Park, Bali 168 C195212 Cultural Park, Bali 71 1952213CR Cultural Park, Bali 169 C195311 Cultural Park, Bali 72 195222CR Cultural Park, Bali 170 C195321A Cultural Park, Bali 73 195224CR Cultural Park, Bali 171 C196921 Botanical Garden, Bali 74 195226CR Cultural Park, Bali 172 CA1-BKBSO Beach, West Kalimantan 75 195227CR Cultural Park, Bali 173 CA2-SO12 Mangrove, Jakarta 76 195227GnCR Cultural Park, Bali 174 DHE 2-1 Enggano Island, Bengkulu 77 195228CR Cultural Park, Bali 175 DHE 9-4 Enggano Island, Bengkulu 78 195231CR Cultural Park, Bali 176 GBSL-9 Lampung, Indonesia 79 195232CR Cultural Park, Bali 177 GKRL-2 Lampung, Indonesia 80 195233CR Cultural Park, Bali 178 GKRL-3 Lampung, Indonesia 81 195321BCR Cultural Park, Bali 179 GKRL-4 Lampung, Indonesia 82 195331CR Cultural Park, Bali 180 MAE 1-11 Enggano Island, Bengkulu 83 195332CR Cultural Park, Bali 181 MAE 1-3 Enggano Island, Bengkulu 84 195333CR Cultural Park, Bali 182 SHP 1-2 Enggano Island, Bengkulu 85 195334CR Cultural Park, Bali 183 SHP 1-4 Enggano Island, Bengkulu 86 195335CR Cultural Park, Bali 184 SHP 1-5 Enggano Island, Bengkulu 87 195336CR Cultural Park, Bali 185 SHP 1-6 Enggano Island, Bengkulu 88 195337CR Cultural Park, Bali 186 SHP 2-2 Enggano Island, Bengkulu 89 195338CR Cultural Park, Bali 187 SHP 2-4 Enggano Island, Bengkulu 90 195339CR Cultural Park, Bali 188 SHP 2-5 Enggano Island, Bengkulu 91 1965114CR Botanical Garden, 189 SHP 6-2 Enggano Island, Bali Bengkulu 92 196511CR Botanical Garden, 190 SHP 6-3 Enggano Island, Bali Bengkulu 93 196512CR Botanical Garden, 191 SHP 6-4 Enggano Island, Bali Bengkulu

110

7 Appendix

94 196522CR Botanical Garden, 192 SHP 6-5 Enggano Island, Bali Bengkulu 95 196523CR Botanical Garden, 193 SHP 6-6 Enggano Island, Bali Bengkulu 96 196524CR Botanical Garden, 194 SHP 7-1 Enggano Island, Bali Bengkulu 97 196526CR Botanical Garden, 195 SHP 7-3 Enggano Island, Bali Bengkulu 98 196532CR Botanical Garden, 196 SHP 7-5 Enggano Island, Bali Bengkulu

Table S 2. List of isolated Actinobacteria with the closest species based on 16S rRNA gene analysis

No. Name of Strain Closest type strain by 16S Similarity Completeness of rRNA gene analysis (%) the sequence (%) Streptomyces strain 1 9BLSSO Streptomyces lanatus 97.03 99.9 2 195105 Streptomyces cyaneus 98.77 50.7 3 195107 Streptomyces cyaneus 98.77 50.6 4 195227GnCR Streptomyces filipinensis 98.81 58.0 5 DHE 9-4 Streptomyces spongiae 98.83 100 6 190122BCR Streptomyces roietensis 98.89 58.2 7 190233 Streptomyces glomeratus 98.96 33.3 8 SHP 1-2 Streptomyces 99.03 99.9 viridochromogenes 9 198414CR Streptomyces cinnabarigriseus 99.14 64.5 10 GKRL-2 Streptomyces jiujiangensis 99.17 100 11 198333CR Streptomyces 99.37 55.1 griseochromogenes 12 C194911 Streptomyces spiralis 99.40 58.2 13 SHP 6-4 Streptomyces xylanilyticus 99.45 100 14 194601 Streptomyces viridiviolaceus 99.45 50.1 15 GBSL-9 Streptomyces jiujiangensis 99.48 66.3 16 1965114CR Streptomyces pharetrae 99.49 54.5 17 195337CR Streptomyces cadmiisoli 99.51 56.3 18 196932CR Streptomyces canus 99.52 58.0 19 190234 Streptomyces misionensis 99.52 28.7 20 197019CR Streptomyces antibioticus 99.55 61.7 21 190235 Streptomyces rhizosphaericus 99.61 53.6 22 C194922A Streptomyces prasinosporus 99.63 36.9 23 C195311 Streptomyces olivaceus 99.71 47.5 24 190224CR Streptomyces lannensis 99.72 48.7 25 195003R Streptomyces tuirus 99.75 55.9 26 198332CR Streptomyces gilvifuscus 99.75 55.1 27 198335CR Streptomyces panaciradicis 99.82 38.5 28 1951016 Streptomyces xiangtanensis 99.86 50.6 29 190232 Streptomyces jiujiangensis 99.88 58.2

111

7 Appendix

30 195213BCR Streptomyces indiaensis 99.88 58.2 31 GKRL-3 Streptomyces hyaluromycini 99.88 56.0 32 GKRL-4 Streptomyces hyaluromycini 99.89 64.0 33 4433 Streptomyces albogriseolus 100 58.6 34 190221 Streptomyces lannensis 100 48.7 35 190224 Streptomyces katrae 100 29.0 36 190225 Streptomyces lucensis 100 30.3 37 190231 Streptomyces lannensis 100 29.3 38 180822CR Streptomyces 100 61.1 griseorubiginosus 39 194813CR Streptomyces levis 100 58.2 40 194823CR Streptomyces levis 100 46.6 41 196511CR Streptomyces 100 55.1 griseorubiginosus 42 196512CR Streptomyces antibioticus 100 58.8 43 2122-5 Streptomyces badius 100 52.3 44 C195321A Streptomyces levis 100 54.9 45 C196921 Streptomyces violaceolatus 100 61.7 46 MAE 1-3 Streptomyces albogriseolus 100 52.9 47 SHP 1-4 Streptomyces hydrogenans 100 61.8 48 SHP 1-5 Streptomyces hydrogenans 100 48.7 49 SHP 1-6 Streptomyces hydrogenans 100 59.4 50 SHP 2-2 Streptomyces hydrogenans 100 29.9 51 SHP 2-4 Streptomyces hydrogenans 100 61.8 52 SHP 2-5 Streptomyces olivaceus 100 45.1 53 SHP 6-2 Streptomyces hydrogenans 100 57.8 54 SHP 6-3 Streptomyces althioticus 100 42.5 55 SHP 6-5 Streptomyces hydrogenans 100 61.8 56 SHP 6-6 Streptomyces hydrogenans 100 53.5 57 SHP 7-1 Streptomyces albidoflavus 100 68 58 SHP 7-3 Streptomyces olivaceus 100 42.2 59 SHP 7-5 Streptomyces hydrogenans 100 48.7 Non-Streptomyces strain 1 195336CR Mycobacterium palauense 98.47 100 2 194938CR Kibdelosporangium 98.74 100 banguiense 3 196526CR Amycolatopsis thermalba 98.85 100 4 2118-1 Nocardia rhizosphaerae 99.13 40 5 195334CR Amycolatopsis magusensis 99.37 100 6 190401 Microbispora hainanensis 99.38 44.7 7 195232CR Couchioplanes caeruleus 99.62 55.2 subsp. Caeruleus 8 4431 Nocardiopsis lucentensis 99.78 62.2 9 4422 Nocardiopsis lucentensis 99.83 39.4 10 180824CR Amycolatopsis japonica 99.80 35.5 11 4421 Nocardiopsis lucentensis 99.86 49.3 12 4423 Nocardiopsis lucentensis 99.86 49.3

112

7 Appendix

13 4435 Nocardiopsis lucentensis 99.87 54.0 14 1982-4 Nocardia araoensis 99.87 52.7 15 1982-1 Nocardia araoensis 99.87 52.5 16 180813CR Nonomuraea endophytica 99.89 61.5 17 MAE 1-11 Kitasatospora albolonga 99.93 100 18 1808210CR Nonomuraea endophytica 100 58.9 19 5931 Pseudonocardia kongjuensis 100 63.6 20 1982-3 Micromonospora 100 59.8 wenchangensis 21 4-5BKBSO Pseudonocardia 100 62.2 carboxydivorans 22 BLH 12-3 Micromonospora aurantiaca 100 29.9 23 C190221 Kitasatospora putterlickiae 100 46.6 24 C1950A Saccharothrix xinjiangensis 100 29.2 25 DHE 2-1 Kitasatospora albolonga 100 59.0

Table S 3. List of strains that can produce extracts with moderate and strong activity against some microbes

No. Strain Production Antimicrobial activity medium 1 C194911 5294 Gram-negative bacteria (Streptomyces sp.) Escherichia coli (moderate) Escherichia coli TolC (moderate) Chromobacterium violaceum (strong) Gram-positive bacteria Bacillus subtilis (strong) Staphylococcus aureus (strong) Micrococcus luteus (strong)

Fungi Mucor hiemalis (strong) Pichia anomala (strong) Candida albicans (strong) 2 195003R 5294 Gram-negative bacteria (Streptomyces sp.) Escherichia coli TolC (moderate) Chromobacterium violaceum (moderate) Gram-positive bacteria Bacillus subtilis (strong) Staphylococcus aureus (strong) Micrococcus luteus (strong) Mycobacterium smegmatis (strong) Fungi Mucor hiemalis (moderate) 3 1951016 5294 Gram-negative bacteria (Streptomyces sp.) Escherichia coli TolC (moderate) Gram-positive bacteria Micrococcus luteus (moderate)

113

7 Appendix

Mycobacterium smegmatis (moderate)

Fungi Candida albicans (moderate) 4 196512CR 5254 Gram-negative bacteria (Streptomyces sp.) Chromobacterium violaceum (moderate) Gram-positive bacteria Bacillus subtilis (strong) Staphylococcus aureus (strong) Micrococcus luteus (strong) Mycobacterium smegmatis (moderate)

Fungi Mucor hiemalis (moderate) Pichia anomala (moderate) 5 C196921 5254 Gram-negative bacteria (Streptomyces sp.) Escherichia coli TolC (moderate) Gram-positive bacteria Bacillus subtilis (moderate) Staphylococcus aureus (moderate) Fungi Candida albicans (moderate) 6 4421 5254+SW Gram-negative bacteria (Nocardiopsis sp.) Escherichia coli TolC (moderate) Gram-positive bacteria Staphylococcus aureus (moderate) Mycobacterium smegmatis (moderate) Bacillus subtilis (strong) Micrococcus luteus (strong) 7 4423 5294+SW Gram-negative bacteria (Nocardiopsis sp.) Escherichia coli TolC (moderate) Gram-positive bacteria Staphylococcus aureus (moderate) Mycobacterium smegmatis (moderate) Bacillus subtilis (strong) Micrococcus luteus (strong) 8 4431 5254 Gram-negative bacteria (Nocardiopsis sp.) Escherichia coli TolC (moderate) Gram-positive bacteria Micrococcus luteus (moderate) 9 180822CR 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) Staphylococcus aureus (strong) 10 195232CR 5294 Gram-negative bacteria (Couchioplanes sp.) Escherichia coli TolC (moderate) Gram-positive bacteria Staphylococcus aureus (moderate)

114

7 Appendix

Mycobacterium smegmatis (moderate)

11 194938CR 5254 Gram-negative bacteria (Kibdelosporangium sp.) Escherichia coli TolC (moderate) Gram-positive bacteria Staphylococcus aureus (moderate) 12 C195212 5254 Gram-negative bacteria Escherichia coli TolC (moderate) Chromobacterium violaceum (moderate)

Gram-positive bacteria Bacillus subtilis (strong) Micrococcus luteus (moderate) 13 SHP 6-3 5294 Gram-negative bacteria (Streptomyces sp.) Escherichia coli TolC (moderate) Gram-positive bacteria Micrococcus luteus (moderate) 14 190224 5254 Gram-positive bacteria (Streptomyces sp.) Micrococcus luteus (moderate) Fungi Mucor hiemalis (moderate) Pichia anomala (moderate) 15 190235 5294 Gram-positive bacteria (Streptomyces sp.) Staphylococcus aureus (strong) Bacillus subtilis (strong) Micrococcus luteus (strong) Fungi Mucor hiemalis (strong) Pichia anomala (strong) Candida albicans (strong) 16 195105 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) Staphylococcus aureus (moderate) Micrococcus luteus (moderate) Mycobacterium smegmatis (moderate) Fungi Mucor hiemalis (moderate) 17 195107 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) Staphylococcus aureus (strong) Micrococcus luteus (moderate) Mycobacterium smegmatis (moderate) Fungi Mucor hiemalis (moderate) 18 195334CR 5294 Gram-positive bacteria (Amycolatopsis sp.) Staphylococcus aureus (moderate) Mycobacterium smegmatis (moderate)

115

7 Appendix

Fungi Mucor hiemalis (moderate) 19 195337CR 5254 Gram-positive bacteria (Streptomyces sp.) Staphylococcus aureus (strong) Fungi Mucor hiemalis (moderate) 20 196526CR 5294 Gram-positive bacteria (Amycolatopsis sp.) Bacillus subtilis (moderate) Staphylococcus aureus (moderate) Fungi Mucor hiemalis (moderate) Candida albicans (moderate)

21 197019CR 5254 Gram-positive bacteria (Streptomyces sp.) Staphylococcus aureus (moderate) Fungi Mucor hiemalis (strong) 22 C194922A 5254 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (moderate) Micrococcus luteus (moderate) Fungi Mucor hiemalis (moderate) 23 C1950A 5294 Gram-positive bacteria (Saccharothrix sp.) Bacillus subtilis (moderate) Staphylococcus aureus (moderate) Micrococcus luteus (moderate)

Fungi Mucor hiemalis (moderate) 24 9BLSSO 5294 Gram-positive bacteria (Streptomyces sp.) Staphylococcus aureus (moderate) Fungi Mucor hiemalis (moderate) 25 SHP 1-4 5254 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (moderate) Fungi Pichia anomala (moderate) 26 SHP 6-6 5254 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (moderate) Fungi Pichia anomala (moderate) 27 4422 5294+SW Gram-positive bacteria (Nocardiopsis sp.) Staphylococcus aureus (moderate) Bacillus subtilis (strong) Micrococcus luteus (strong) 28 SHP 1-2 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) Staphylococcus aureus (moderate)

116

7 Appendix

Micrococcus luteus (strong) 29 4435 5294+SW Gram-positive bacteria (Nocardiopsis sp.) Micrococcus luteus (moderate) Bacillus subtilis (strong) 30 190231 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (moderate) Micrococcus luteus (moderate) 31 190234 5294 Gram-positive bacteria (Streptomyces sp.) Micrococcus luteus (moderate) 32 180824CR 5294 Gram-positive bacteria (Amycolatopsis sp.) Bacillus subtilis (moderate) 33 194823CR 5254 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (moderate) Staphylococcus aureus (moderate) 34 194921CR 5294 Gram-positive bacteria Staphylococcus aureus (strong) 35 195211CR 5294 Gram-positive bacteria Staphylococcus aureus (strong) 36 195213BCR 5294 Gram-positive bacteria (Streptomyces sp.) Staphylococcus aureus (moderate) 37 195231CR 5294 Gram-positive bacteria Staphylococcus aureus (moderate) 38 198335CR 5254 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) 39 SHP 2-5 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) Micrococcus luteus (strong)

40 SHP 6-4 5294 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (moderate) Staphylococcus aureus (moderate) 41 SHP 7-3 5254 Gram-positive bacteria (Streptomyces sp.) Bacillus subtilis (strong) Micrococcus luteus (strong)

42 190232 5294 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) 43 190233 5294 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) 44 194601 5294 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) Pichia anomala (moderate) 45 190224CR 5254 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) 46 195227GnCR 5254 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) 47 196932CR 5254 Fungi (Streptomyces sp.) Mucor hiemalis (moderate)

117

7 Appendix

48 198332CR 5254 Fungi (Streptomyces sp.) Mucor hiemalis (strong) Candida albicans (strong)

49 4-5BKBSO 5294 Fungi (Pseudonocardia sp.) Mucor hiemalis (moderate) 50 C190221 5294 Fungi (Kitasatospora sp.) Pichia anomala (moderate) Candida albicans (moderate) 51 C195321A 5294 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) Pichia anomala (moderate) 52 DHE 2-1 5254 Fungi (Kitasatospora sp.) Mucor hiemalis (strong) Pichia anomala (moderate) 53 SHP 1-6 5254 Fungi (Streptomyces sp.) Pichia anomala (moderate) 54 SHP 2-2 5254 Fungi (Streptomyces sp.) Pichia anomala (moderate) 55 SHP 2-4 5254 Fungi (Streptomyces sp.) Pichia anomala (moderate) 56 SHP 6-5 5254 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) Pichia anomala (moderate) 57 SHP 7-1 5254 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) 58 SHP 7-5 5254 Fungi (Streptomyces sp.) Mucor hiemalis (moderate) Pichia anomala (moderate)

Table S 4. List of nontoxic extracts having antiviral activity with moderate and strong level against HCV

No. Strain Extract Antiviral activity level 1 DHE 2-1 DHE 2-1_5254 Very Strong 2 DHE 2-1 DHE 2-1_5294 Very Strong 3 MAE 1-11 MAE 1-11_5294 Very Strong 4 MAE 1-11 MAE 1-11_SYP Very Strong 5 SHP 1-4 SHP 1-4_5294 Very Strong 6 SHP 2-2 SHP 2-2_5294 Very Strong 7 SHP 6-6 SHP 6-6_5294 Very Strong 8 190231 190231_5294 Very Strong 9 C190221 C190221_5254 Very Strong 10 C194911 C194911_5294 Very Strong

118

7 Appendix

11 C195321A C195321A_5254 Very Strong 12 C195321A C195321A_5294 Very Strong 13 C196921 C19692 _5254 Very Strong 14 4421 4421_5254 Strong 15 4421 4421_5254+SW Strong 16 4423 4423_5254 Strong 17 4423 4423_5254+SW Strong 18 4435 4435_5254+SW Strong 19 5931 5931_5254+SW Strong 20 5931 5931_5294+SW Strong 21 SHP 1-5 SHP 1-5_5294 Strong 22 SHP 1-6 SHP 1-6_5294 Strong 23 SHP 6-2 SHP 6-2_5254 Strong 24 SHP 6-2 SHP 6-2_5294 Strong 25 SHP 7-5 SHP 7-5_5294 Strong 26 190401 190401_5254 Strong 27 C194911 C194911_5254 Strong 28 C194922A C194922A _5254 Strong 29 4422 4422_5254 Moderate 30 4422 4422_5254+SW Moderate 31 4423 4423_5294 Moderate 32 4431 4431_5254 Moderate 33 4431 4431_5294 Moderate 34 4431 4431_5254+SW Moderate 35 4433 4433_5254+SW Moderate 36 4435 4435_5294+SW Moderate 37 5931 5931_5254 Moderate 38 BLH 12-3 BLH 12-3_5254 Moderate 39 BLH 12-3 BLH 12-3_5294 Moderate 40 DHE 9-4 DHE 9-4_5254 Moderate 41 DHE 9-4 DHE 9-4_5294 Moderate 42 SHP 1-6 SHP 1-6_5254 Moderate 43 SHP 6-5 SHP 6-5_5294 Moderate 44 C195311 C195311_5254 Moderate 45 C196921 C196921_5294 Moderate 46 C194922A C194922A _5294 Moderate

119

7 Appendix

Table S 5. Phenotypic comparison between Streptomyces sp. SHP 1-2, Streptomyces fumigatiscleroticus DSM 43154 T, Streptomyces malachitofuscus DSM 40332T, Streptomyces viridochromogenes DSM 40110 T, and Streptomyces misionensis DSM 40306.

Characteristic 1 2 3 4 5 ISP 2 - growth Good Good Good Good Sparse ISP 2 - colony color Olive brown, Yellow Ochre Green Green ochre yellow brown brown ISP 2 - aerial mycelium Traffic grey B, Sparse Grey Blue None signal white grey ISP 2 - soluble pigment None None None None None ISP 3 - growth Good Good Good Good Good ISP 3 - colony color Ivory Yellow/ Yellow Green Beige Brown grey ISP 3 - aerial mycelium Telegrey 2, Sparse White Blue Beige signal white grey grey ISP 3 - soluble pigment None None None Green None ISP 4 - growth Moderate Good Good Good Sparse ISP 4 - colony color Ivory Colorless Colorless Green Olive brown ISP 4 - aerial mycelium Telegrey 2, White Sparse, Blue None signal white white grey ISP 4 - soluble pigment None None None None None ISP 5 - growth Good Good Good Good Good ISP 5 - colony color Ivory, light Red Yellow Green Beige ivory brown grey ISP 5 - aerial mycelium Telegrey 2, White White Blue Signal signal white grey white ISP 5 - soluble pigment None None None Brown None ISP 6 - growth Good Good Good Good Sparse ISP 6 - colony color Maize yellow Colorless Brown Green Beige grey ISP 6 - aerial mycelium Signal white None None Blue None grey ISP 6 - soluble pigment None None Brown Brown None ISP 7 - growth Good Good Good Good Sparse ISP 7 - colony color Ivory light, Red Black Green Beige ivory brown grey ISP 7 - aerial mycelium Traffic grey A, Grey White Blue Dusty signal white grey grey ISP 7 - soluble pigment None Red Black None None Use of carbohydrate Glucose + - + + + Arabinose - + + + (+) Sucrose - - + - - Xylose - - - + ++ Inositol - - + + (+) Mannitol - + (+) + (+) Fructose - + + + (+) Rhamnose - + + - -

120

7 Appendix

Raffinose - + - - - Cellulose - - - - - API ZYM

Phosphatase alcaline ++ ++ ++ ++ ++ Esterase (C4) (+) + (+) + + Esterase Lipase (C8) (+) (+) (+) + ++ Lipase (C14) (+) - - - (+) Leucin arylamidase ++ ++ ++ ++ ++ Valine arylamidase (+) ++ + + ++ Cystine arylamidase (+) + + + + Trypsin - (+) + + - Chymotrypsin - (+) + (+) (+) Phosphatase acid (+) ++ ++ ++ ++ Naphtol-AS-BI-phosphohydrolase (+) ++ ++ ++ ++ α-Galactosidase - (+) - - + β-Galactosidase (+) ++ + ++ ++ β-Glucuronidase - - - - - α-Glucosidase ++ + ++ - ++ β-Glucosidase (+) ++ (+) ++ - N-acetyl-beta-glucoseamidase ++ - + ++ ++ α-Mannosidase - ++ ++ ++ ++ α-Fucosidase - - - - - API Coryne Nitrate reduction - + - - - Pyraziamidase - - - - - Pyrrolidonyl arylamidase - - - - + Alkaline phosphatase + + + + + β-Glucuronidase - - - - - β-Galactosidase - + - - + α-Glucosidase + + + - + N-acetyl -beta glucoseamidase + + - + + Esculin (beta glucosidase) + + - - + Urease - - - - - Gelatine(hydrolysis) + + + - + Glucose fermentation - - - - - Ribose fermentation - - - - - Xylose fermentation - - - - - Mannitol fermentation - - - - - Maltose fermentation - - - - - Lactose fermentation - - - - - Sucrose fermentation - - - - - Glycogen fermentation - - - - - ++ more positive result; + positive result; - negative result; (+) weakly positive result; 1: Strain SHP 1- 2; 2: Streptomyces fumigatiscleroticus DSM 43154 T; 3: Streptomyces malachitofuscus DSM 40332; 4: Streptomyces viridochromogenes DSM 40110; 5: Streptomyces misionensis DSM 40306.

121

7 Appendix

Figure S 1. Chromatogram and spectra of Indolactam variant 1.

122

7 Appendix

Figure S 2. Chromatogram and spectra of Indolactam variant 2.

123

7 Appendix

Table S 6. NMR data of indolactam variant 1

No No. C Shift XHn H Shift H Multiplicity COSY N/ROESY H to C HMBC C to H HMBC TOCSY H Mark Atom 1 19 16.852 CH3 0.965 d (6.45) 2.57 2.57, 2.99, 3.47, 2.57, 2.99, 3.47, 37.25, 64.70, 68.56 2.57, 2.99, 4.68 4.68 3.47, 4.68 2 20 33.947 CH3 2.897 s 2.57, 2.99, 3.47, 4.68 68.56, 106.58, 4.15, 6.49 148.43 3 10 35.514 CH2 3.082 dd (17.21, 3.47, 3.67, 4.68, 3.47, 7.03 65.76, 119.71 7.03 '' 3.76) 7.03 4 10 35.514 CH2 3.107 m 7.03 3.67, 4.15, 4.68, 3.47, 7.03 57.11 3.47, 7.03 ' 6.35, 7.03 5 17 37.246 CH 2.567 dqdt (10.10, 0.96, 2.99, 0.96, 2.90, 2.99, 0.96, 4.68 16.85, 68.56, 0.96, 2.99, 6.45, 6.45, 4.68 3.47, 3.47, 4.68 172.95 3.47, 3.47, 6.45, 4.00, 4.68 3.23, 3.23) 6 11 57.112 CH 4.147 dqd (9.00, 3.09, 3.47, 2.90, 3.11, 3.47, 3.11, 3.47 3.09, 3.47, 4.40, 4.40, 3.67 3.67, 4.68, 6.35 3.67, 6.35 4.30, 3.87) 7 18 64.701 CH2 3.470 dd (10.54, 2.99 0.96, 2.57, 2.90, 0.96, 4.68 16.85, 68.56 0.96, 2.57, '' 3.00) 6.49 4.68 8 18 64.701 CH2 2.987 dd (10.54, 2.57, 3.47 0.96, 2.57, 2.90, 0.96, 4.68 16.85 0.96, 2.57, ' 4.20) 3.47, 4.68, 6.49 4.68 9 16 65.758 CH2 3.475 dd (11.00, 3.67, 4.15 2.57, 2.99, 3.08, 3.08 35.51, 57.11 2.57, 3.09, 9.00) 3.67, 4.15, 4.68, 3.11, 3.67, 6.35 4.15, 6.35 10 16 65.758 CH2 3.666 dd (11.00, 3.47, 4.15 3.08, 3.11, 3.47, 3.08 3.09, 3.47, 4.41) 4.15, 6.35 4.15, 6.35 11 14 68.556 CH 4.682 d (10.11) 2.57 0.96, 2.57, 2.99, 0.96, 2.57, 2.90, 16.85, 33.95, 0.96, 2.57, 3.08, 3.11, 3.47, 3.47 37.25, 64.70, 2.99, 3.47 4.15 148.43, 172.95 12 8 106.061 CH 6.934 d (8.00) 10.05 6.49, 106.58 106.58, 119.71 6.49

13 6 106.581 CH 6.488 d (7.53) 6.97 2.90, 2.99, 3.47 2.90, 6.93, 6.97, 106.06, 115.31, 6.93 106.06 119.71 14 3 115.314 C 3.09, 6.49, 7.03

124

7 Appendix

15 4 119.715 C 3.08, 6.49, 6.93, 6.97, 7.03 16 123.128 C

17 2 123.492 CH 7.035 s 3.11 3.08, 3.11, 10.05 3.09 35.51, 115.31, 3.08, 3.11, 119.71 10.05 18 9 141.131 C 6.97

19 5 148.435 C 2.90, 4.68, 6.97

20 13 172.953 C 2.57, 4.68

21 3.093 m 4.15 115.31, 123.49 3.47, 3.67, 4.15 22 12 NH 6.353 br s 3.11, 3.47, 3.67, 3.47, 3.67, 4.15 4.15 23 1 NH 10.046 br s 6.93, 7.03 7.03

24 7 CH 6.973 dd (8.00, 7.53) 6.49 106.58, 119.71, 141.13, 148.43

Table S 7. NMR data of indolactam variant 2

No Atom No. C Shift XHn H Shift H Multiplicity COSY N/ROESY H to C HMBC C to H HMBC TOCSY H Mark 1 19 15.570 CH3 0.676 d (7.10) 2.58 2.58, 2.88, 3.44, 2.58, 3.44, 3.58, 37.45, 67.20, 2.58, 3.44, 3.58, 4.92, 6.44 4.92 173.96 3.58, 4.92 2 20 33.720 CH3 2.879 s 0.68, 2.58, 4.15, 4.92 67.20, 106.68, 4.92, 6.44 149.15 3 10 35.072 CH2 3.199 br dd (17.21, 3.01, 4.15, 3.01, 3.66, 4.15, 3.47, 3.66, 7.00 57.20, 65.73, 3.01, 3.47, ' 4.09) 7.00 4.92, 7.00 115.68, 123.09 3.66, 4.15, 7.00 4 10 35.072 CH2 3.013 dd (17.21, 3.20, 4.15 3.20, 3.47, 3.66, 3.47, 3.66, 7.00 57.20, 65.73, 3.20, 3.47, '' 3.76) 4.15, 7.00 115.68, 119.62, 3.66, 4.15, 123.09 7.00 5 17 37.446 CH 2.579 dqdd (10.40, 0.68, 3.44, 0.68, 2.88, 3.44, 0.68, 3.44, 3.58, 15.57, 67.20, 0.68, 3.44, 7.10, 7.10, 3.58, 4.92 3.58, 4.15, 4.92 4.92 173.96 3.58, 4.92 7.10, 3.66, 3.23)

125

7 Appendix

6 11 57.199 CH 4.150 ddddd (9.40, 3.01, 3.20, 2.58, 2.88, 3.01, 3.01, 3.20, 3.47 65.73, 115.68 3.01, 3.20, 4.52, 4.40, 3.47, 3.66 3.20, 3.47, 3.66, 3.47, 3.66 4.09, 3.76) 4.92, 6.37 7 16 65.732 CH2 3.661 dd (11.19, 3.47, 4.15 3.01, 3.20, 3.47, 3.01, 3.20, 3.38, 35.07 3.01, 3.20, 4.52) 3.58, 4.15, 6.37 4.15 3.47, 4.15 8 16 65.732 CH2 3.467 dd (11.00, 3.66, 4.15 3.01, 3.58, 3.66, 3.01, 3.20, 3.38, 35.07, 57.20 3.01, 3.20, 9.25) 4.15, 6.37 4.15 3.66, 4.15 9 18 66.469 CH2 3.581 dd (10.54, 2.58, 3.44 0.68, 2.58, 3.44, 4.92 15.57, 37.45 0.68, 2.58, 3.66) 3.47, 3.66, 4.92 3.44, 4.92 10 18 66.469 CH2 3.442 dd (10.54, 2.58, 3.58 0.68, 2.58, 3.58, 4.92 15.57, 37.45, 67.20 0.68, 2.58, 3.23) 4.92 3.58, 4.92 11 14 67.205 CH 4.919 d (10.40) 2.58 0.68, 2.58, 2.88, 0.68, 2.58, 2.88, 15.57, 33.72, 0.68, 2.58, 3.20, 3.44, 3.58, 3.44, 6.44 37.45, 66.47, 3.44, 3.58 4.15 149.15, 173.96 12 8 105.567 CH 6.897 dt (8.00, 0.80, 9.98 6.44 106.68, 119.62, 6.44 0.80) 149.15 13 6 106.676 CH 6.440 d (7.53) 6.95 0.68, 2.88 2.88, 6.90, 6.95 67.20, 105.57, 6.90 115.68, 119.62, 149.15 14 3 115.678 C 3.01, 3.20, 4.15, 6.44, 7.00 15 4 119.620 C 3.01, 6.44, 6.90, 7.00 16 2 123.094 CH 7.003 d (1.29) 3.20 3.01, 3.20, 9.98 3.01, 3.20 35.07, 115.68, 3.01, 3.20 119.62, 141.24 17 7 123.406 CH 6.948 dd (8.00, 7.53) 6.44 106.68, 141.24, 149.15 18 9 141.235 C 6.95, 7.00 19 5 149.154 C 2.88, 4.92, 6.44, 6.90, 6.95 20 13 173.958 C 0.68, 2.58, 4.92 21 1 NH 9.981 br s 6.90, 7.00 24 12 NH 6.369 br s 3.47, 3.66, 4.15

126

7 Appendix

Table S 8. NMR data of nitrososaxin C

a Nitrososaxin C (CDCl3) 6526-61-63 (CDCl3) 1H-NMR 13C-NMR 1H-NMR 13C-NMR 0.86 (6H, d, 6.85 Hz) 22.63 0.87 (6H, d, 6.45) 22.64 1.13-1.32 (18H, m) 26.12 1.14-1.27 (18H, m) 27.10 1.51 (1H, m) 26.59 1.52 (1H, dt, 13.28 Hz, 6.59 Hz) 27.11 1.94 (2H, q, 7.26 Hz) 27.38 1.71-1.81 (2H, br, s) 27.41 4.13 (2H, t, 7.26 Hz) 27.95 27.96 11.52 (1H, s) 28.81 29.11 29.27 29.29 29.45 29.46 29.55 29.57 29.64 29.64 29.89 29.93 39.04 39.04 61.38 65.12b aNishio et al., 1993226; bdetermined in HSQC-DEPT

127